Ion exchange chromatography with improved selectivity for the separation of polypeptide monomers, aggregates and fragments by modulation of the mobile phase

ABSTRACT

Herein is reported a method for producing a polypeptide in monomeric form comprising the following step: recovering the polypeptide in monomeric form from an ion exchange chromatography material by applying a solution comprising a non-ionic polymer and an additive.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2012/054449 having an international filing date of Mar. 14, 2012,the entire contents of which are incorporated herein by reference, andwhich claims benefit under 35 U.S.C. §119 to European Patent ApplicationNo. 11158523.8 filed Mar. 16, 2011.

Herein is reported a method for separating polypeptide monomers,aggregates and fragments using ion exchange chromatography, whereby inthe recovering step a non-ionic polymer and an additive are added to themobile phase.

BACKGROUND OF THE INVENTION

Proteins and especially immunoglobulins play an important role intoday's medical portfolio. Polypeptides for use in pharmaceuticalapplications are mainly produced in mammalian cells such as CHO cells,NS0 cells, Sp2/0 cells, COS cells, HEK cells, BHK cells, PER.C6® cells,and the like.

Due to their chemical and physical properties, such as molecular weightand domain architecture including secondary modifications, thedownstream processing of immunoglobulins is very complicated. Forexample, are not only for formulated drugs but also for intermediates indownstream processing (DSP) concentrated solutions required to achievelow volumes for economic handling and application storage. Thedownstream processing of biotechnologically produced immunoglobulinstypically comprises three chromatography steps: a first affinitychromatography step using e.g. Protein A, to remove non-immunoglobulinmolecules, followed by two ion exchange chromatography steps. Thepurified immunoglobulin is obtained in a low concentration solutionrequiring a concentration step prior to formulating the antibody intothe pharmaceutical formulation. Many proteins, including IgG, formdimers, oligomers or higher order aggregates. In order to provide atherapeutic protein product with the required purity these moleculespecies have to be removed by the purification process.

Different methods are well established and widely used for proteinpurification (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998)93-102). Zhou, J. X., et al. (J. Chrom. A 1175 (2007) 69-80) report apH-conductivity hybrid gradient cation-exchange chromatography forprocess-scale monoclonal antibody purification. The separation ofantibody monomers from its multimers by use of ion exchangechromatography is reported in EP 1 084 136. U.S. Pat. No. 5,429,746relates to the application of hydrophobic interaction chromatographycombination chromatography to the purification of antibody moleculeproteins.

Solvent modulation of column chromatography is reported by Arakawa, T.,et al. in Prot. Pep. Lett. 15 (2008) 544-555. In EP 1 729 867 a methodfor chromatographic purification is reported. Antibody aggregate removalby hydroxyapatite chromatography is reported by Gagnon, P. and Beam, K.,in Curr. Pharm. Biotechnol. 10 (2009) 440-446. In J. Immunol. Meth. (336(2008) 222-228) reports Gagnon, P., an improved antibody aggregateremoval by hydroxyapatite chromatography in the presence of polyethyleneglycol. In WO 2004/013162 an increased dynamic binding capacity in ionexchange chromatography by addition of polyethylene glycol is reported.In WO 2005/094960 a method for chromatographic purification is reported.Enhanced capacity and purification of antibodies by mixed modechromatography in the presence of aqueous-soluble non-ionic organicpolymers is reported in WO 2008/086335. In WO 2009/149067 a process forthe purification of antibodies is reported.

Milby, K. H., et al. report the ion-exchange chromatography of proteinsand the effect of neutral polymers in the mobile phase (J. Chrom. 482(1989) 133-144). In Biotechnology Techniques (12 (1998) 289-293) Feng,X-L., et al. report that polyethylene glycol improves the purificationof recombinant human tumor necrosis factor during ion exchangechromatography. A method for obtaining unique selectivities inion-exchange chromatography by addition of organic polymers to themobile phase is reported by Gagnon, P., et al. (J. Chrom. 743 (1996)51-55). Gagnon, P., reports in an internet article on validated.com(http://www.validated.com/revalbio/pdffiles/ionslect.pdf) thefine-tuning of selectivity on ion exchangers. In WO 2009/149067 aprocess for the purification of antibodies is reported.

SUMMARY OF THE INVENTION

It has been found that high PEG-concentrations can be employed duringcation exchange chromatography in the presence of certain additives(solubility enhancers) in order to modulate selectivity and to improveresolution of antibody monomer and aggregate species, e.g. compared tomethods employing PEG-only.

Herein is reported a method for isolating or obtaining or producing apolypeptide of interest in monomeric form from other component(s), suchas the polypeptide of interest in aggregated form, comprising at leastone chromatographic step. In one embodiment the method comprisescontacting a solution comprising the polypeptide of interest with acation-exchange chromatography material wherein the contacting with thechromatography material takes place in the absence of a non-ionicpolymer and an additive, and wherein the recovering takes place in thepresence of a non-ionic polymer and an additive.

It has been found that by using an additional additive in a method asreported herein the effect of the non-ionic polymer can be improved.

One aspect as reported herein is a method for producing an antibody inmonomeric form comprising the following steps:

-   -   applying a solution comprising a non-ionic polymer and an        additive to a chromatography material to which an antibody had        been adsorbed, whereby the antibody in monomeric form remains        adsorbed to the ion exchange chromatography material, and    -   recovering the antibody in monomeric form from the ion exchange        chromatography material by applying a solution comprising a        non-ionic polymer, an additive, and an elution compound, and        thereby producing the antibody in monomeric form.

Another aspect as reported herein is a method for producing apolypeptide preparation with reduced host cell protein content wherebythe preparation is obtained from a mammalian cell cultivationsupernatant, especially a CHO cell cultivation supernatant, comprisingthe following steps:

-   -   applying a solution comprising a non-ionic polymer and an        additive to a chromatography material to which the polypeptide        had been adsorbed, whereby the polypeptide in monomeric form        remains adsorbed to the ion exchange chromatography material,        and    -   recovering the polypeptide in monomeric form from the ion        exchange chromatography material by applying a solution        comprising a non-ionic polymer, an additive, and an elution        compound, and thereby producing the polypeptide preparation with        reduced host cell protein content.

One aspect as reported herein is a method for determining theconcentration of a non-ionic polymer and an additive for use in an ionexchange chromatography of a polypeptide comprising the following steps:

-   -   determining in solution in the absence of the additive the        concentration of the non-ionic polymer at which less than 50% of        the polypeptide remain in solution,    -   determining in solution in the presence of a concentration of        the non-ionic polymer, which has been determined in the previous        step, the concentration of the additive at which more than 95%        of the polypeptide remain in solution,        thereby determining the concentration of a non-ionic polymer and        an additive.

One aspect as reported herein is a method for producing an antibody inmonomeric form comprising the following step:

-   -   recovering the antibody in monomeric form from an ion exchange        chromatography material by applying a solution comprising a        non-ionic polymer, an additive, and an elution compound, and        thereby producing the antibody in monomeric form,    -   wherein the separation efficiency (resolution) of the        chromatography is enhanced compared to a chromatography in the        absence of the non-ionic polymer, and    -   wherein the employed concentration of the non-ionic polymer        would result in a partial or complete precipitation of the        antibody in the absence of the additive.

In one embodiment the ion exchange chromatography material is a cationexchange chromatography material.

In one embodiment the non-ionic polymer is selected from the groupcomprising poly (ethylene glycol) (PEG), poly (propylene glycol) (PPG),PEG-PPG copolymers, and triblock copolymers composed of poly(oxypropylene) (poly (propylene oxide)) flanked by poly (oxyethylene)(poly (ethylene oxide)). In a further embodiment the non-ionic polymeris poly (ethylene glycol).

In one embodiment the additive is selected from the group comprisingzwitterions, amino acids, urea, urea derivatives, ampholytes, CHAPSO,natural products, sugars, and polyols. In a further embodiment theadditive is sorbitol.

In one embodiment the non-ionic polymer has a concentration of fromabout 5% to 15% by weight and/or the additive has a concentration offrom 3% to 25% by weight.

In one embodiment the polypeptide is an antibody of class IgG, or IgD,or IgE, or IgA. In another embodiment the polypeptide is an antibody ofclass IgG, subclass IgG1, or subclass IgG2, or subclass IgG3, orsubclass IgG4.

In one embodiment the method comprises the following steps:

-   -   applying a first solution that optionally comprises a non-ionic        polymer and an additive to an ion exchange chromatographic        material and thereby equilibrating the ion exchange        chromatography material,    -   applying a buffered solution comprising the polypeptide to the        equilibrated chromatography material and thereby adsorbing the        polypeptide to the chromatography material, whereby the solution        is essentially free of a non-ionic polymer and an additive,    -   applying a buffered solution comprising a non-ionic polymer and        an additive to the chromatography material, whereby the        polypeptide in monomeric form remains adsorbed to the ion        exchange chromatography material, and    -   recovering the polypeptide in monomeric form from the ion        exchange chromatography material by applying a solution        comprising a non-ionic polymer, an additive, and an elution        compound.

In one embodiment the polypeptide is an antibody.

In one embodiment about 40 g polypeptide (antibody) are applied perliter of chromatography material. In one embodiment about 30 gpolypeptide (antibody) are applied per liter of chromatography material.In one embodiment about 20 g polypeptide (antibody) are applied perliter of chromatography material.

DETAILED DESCRIPTION OF THE INVENTION

Herein is reported a method for producing a polypeptide of interest inmonomeric form by separating it from other component(s), such as thepolypeptide of interest in aggregated form, comprising at least onechromatographic step. In one embodiment the method comprises contactinga solution comprising the polypeptide of interest and a cation-exchangechromatography material wherein the contacting with the chromatographymaterial takes place in the absence of a non-ionic polymer and anadditive and wherein the recovering takes place in the presence of anon-ionic polymer and an additive.

It has been found that the presence of a non-ionic polymer and anadditive increases the retention time difference (resolution) of apolypeptide in monomeric form compared to the polypeptide in aggregatedform on a cation exchange chromatography material, thereby enablingnovel selectivity for improved removal of a polypeptide in aggregatedform from the polypeptide in monomeric form.

Especially it has been found that high PEG-concentrations can beemployed during cation exchange chromatography in the presence ofcertain additives (solubility enhancers) in order to modulateselectivity and to improve resolution of antibody monomer and aggregatespecies, e.g. compared to methods employing PEG-only.

Poly (ethylene glycol) is viscous and high viscosities of the runningbuffers in chromatographic applications lead to certain practicallimitations during chromatographic applications.

A chromatographic separation process can be characterized by theresolution of the individual peaks in the elution chromatogram. Theresolution value can be calculated according to Kaltenbrunner, O., etal., Biotechnol. Bioeng. 98 (2007) 201-210, or Grushka, E., Anal. Chem.44 (1972) 1733-1738.

General chromatographic methods and their use are known to a personskilled in the art. See for example, Heftmann, E., (ed.),Chromatography, 5^(th) edition, Part A: Fundamentals and Techniques,Elsevier Science Publishing Company, New York, (1992); Deyl, Z., (ed.),Advanced Chromatographic and Electromigration Methods in Biosciences,Vol. 60, Elsevier Science BV, Amsterdam, The Netherlands, (1998); Poole,C. F., and Poole, S. K., Chromatography Today, Elsevier SciencePublishing Company, New York (1991); Scopes, Protein Purification:Principles and Practice, Springer Verlag (1982); Sambrook, J. et al.,(ed.), Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); orAusubel, F. M. et al., (eds.), Current Protocols in Molecular Biology,John Wiley & Sons, Inc., New York (1998).

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 20 amino acid residues are referred to as “peptides”. A“protein” is a macromolecule comprising one or more polypeptide chainsor at least one polypeptide chain of more than 100 amino acid residues.A polypeptide may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrate groups and other non-peptidicsubstituents may be added to a polypeptide by the cell in which thepolypeptide is produced, and will vary with the type of cell.Polypeptides are defined herein in terms of their amino acid backbonestructures; substituents such as carbohydrate groups are generally notspecified, but may be present nonetheless.

The term “applying to” denotes a partial step of a purification methodin which a solution is brought in contact with a chromatographymaterial. This denotes that either a) the solution is added to achromatographic device in which the chromatography material iscontained, or b) that the chromatography material is added to a solutioncomprising the polypeptide. In case a) the solution passes through thedevice allowing for the adsorption of the substances contained insolution to the chromatography material. Depending on the conditions,such as e.g. pH, conductivity, salt concentration, temperature, and/orflow rate, some substances of the solution adsorb to the chromatographymaterial and other substances can be recovered from the flow-through.The “flow-through” denotes the solution obtained after the passage ofthe device, which may either be the applied solution or a bufferedsolution, which is used to wash the column or to cause elution ofsubstances adsorbed to the chromatography material. In one embodimentthe device is a column or a cassette. In case b) the chromatographymaterial can be added, e.g. as a solid, to the solution, e.g. containingthe substance of interest to be purified, allowing for an interactionbetween the chromatography material and the substances in solution.After the interaction the chromatography material is removed, e.g. byfiltration, and the substance bound to the chromatography material arealso removed therewith from the solution whereas the substances notbound to the chromatography material remain in solution. In oneembodiment about 40 g polypeptide (antibody) are adsorbed per liter ofchromatography material. In one embodiment about 30 g polypeptide(antibody) are adsorbed per liter of chromatography material. In oneembodiment about 20 g polypeptide (antibody) are adsorbed per liter ofchromatography material.

The term “bind-and-elute mode” denotes an operation mode of achromatography step, in which a solution containing a substance ofinterest to be purified is applied to a chromatography material, wherebythe substance of interest binds to the chromatography material. Thus,the substance of interest is retained on the chromatography materialwhereas substances not of interest are removed with the flow-through orthe supernatant. The substance of interest is afterwards recovered fromthe chromatography material in a second step with an elution solution.In one embodiment the method as reported herein is operated inbind-and-elute mode.

The solutions employed in the method as reported herein are crude orbuffered solutions. The term “buffered solution” denotes a solution inwhich changes of pH due to the addition or release of acidic or alkalinesubstances is leveled by the dissolved buffer substance. Any buffersubstance with such properties can be used. Generally pharmaceuticallyacceptable buffers substances are used. In one embodiment the bufferedsolution is selected from a phosphate buffered solution consisting ofphosphoric acid and/or salts thereof, or an acetate buffered solutionconsisting of acetic acid and salts thereof, or a citrate bufferedsolution consisting of citric acid and/or salts thereof, or a morpholinebuffered solution, or a 2-(N-morpholino) ethanesulfonic bufferedsolution, or a histidine buffered solution, or a glycine bufferedsolution, or a tris (hydroxymethyl) aminomethane (TRIS) bufferedsolution. In another embodiment the buffer solution is selected from aphosphate buffered solution, or an acetate buffered solution, or acitrate buffered solution, or a histidine buffered solution. Optionallythe buffered solution may comprise an additional salt, such as e.g.sodium chloride, sodium sulphate, potassium chloride, potassium sulfate,sodium citrate, or potassium citrate.

The terms “continuous elution” and “continuous elution method” denote amethod wherein the conductivity of a solution causing elution, i.e. therecovery of a bound compound from a chromatography material, is changed,i.e. raised or lowered, continuously, i.e. the concentration is changedby a sequence of small steps each not bigger than a change of 2%, or of1% of the concentration of the substance causing elution. In this“continuous elution” one or more conditions, for example the pH, theionic strength, concentration of a salt, and/or the flow of the mobilephase can be changed linearly or exponentially or asymptotically. In oneembodiment the change is linear.

The term “step elution” denotes a method wherein e.g. the concentrationof a substance causing elution, i.e. the recovery of a bound substancefrom a chromatography material, is raised or lowered at once, i.e.directly from one value/level to the next value/level. In this “stepelution” one or more conditions, for example the pH, the ionic strength,concentration of a salt, and/or the flow of a chromatography, can bechanged all at once from a first, e.g. starting, value to a second, e.g.final, value. Thus, the conditions are changed incrementally, i.e.stepwise, in contrast to a linear change.

The term “ion exchange chromatography material” denotes an immobile highmolecular weight matrix that carries covalently bound chargedsubstituents used as stationary phase in ion exchange chromatography.For overall charge neutrality not covalently bound counter ions arebound thereto. The “ion exchange chromatography material” has theability to exchange its not covalently bound counter ions for similarlycharged ions of the surrounding solution. Depending on the charge of itsexchangeable counter ions the “ion exchange resin” is referred to ascation exchange resin or as anion exchange resin. Depending on thenature of the charged group (substituent) the “ion exchange resin” isreferred to as, e.g. in the case of cation exchange resins, sulfonicacid resin (S), or sulfopropyl resin (SP), or carboxymethyl resin (CM).

Different types of ion exchange materials, i.e. stationary phases, areavailable under different names and from a multitude of companies suchas e.g. cation exchange materials Bio-Rex® (e.g. type 70), Chelex® (e.g.type 100), Macro-Prep® (e.g. type CM, High S, 25 S), AGO (e.g. type 50W,MP) all available from BioRad Laboratories, WCX 2 available fromCiphergen, Dowex® MAC-3 available from Dow chemical company, Mustang Cand Mustang S available from Pall Corporation, Cellulose CM (e.g. type23, 52), hyper-D, partisphere available from Whatman plc., Amberlite®IRC (e.g. type 76, 747, 748), Amberlite® GT 73, Toyopearl® (e.g. typeSP, CM, 650M) all available from Tosoh Bioscience GmbH, CM 1500 and CM3000 available from BioChrom Labs, SP-Sepharose™, CM-Sepharose™available from GE Healthcare, Poros resins available from AppliedBiosystems or PerSeptive Biosystems, Asahipak ES (e.g. type 502C), CXpakP, IEC CM (e.g. type 825, 2825, 5025, LG), IEC SP (e.g. type 420N, 825),IEC QA (e.g. type LG, 825) available from Shoko America Inc., 50W cationexchange resin available from Eichrom Technologies Inc. In oneembodiment the cation exchange material is a strong cation exchangematerial such as Macro-Prep® High 5 or 255, or MacroCap SP, orToyopearl® SP 650M, or Source S, or SP Sepharose, or POLYCAT A, or MonoS, or Highscreen SP.

The term “under conditions suitable for binding” and grammaticalequivalents thereof denotes that a substance of interest, e.g. anantibody in monomeric form, binds to a stationary phase when brought incontact with it, e.g. an ion exchange material. This does notnecessarily denote that 100% of the substance of interest is bound butessentially 100% of the substance of interest is bound, i.e. at least50% of the substance of interest is bound, in one embodiment at least75% of the substance of interest is bound, in another embodiment atleast 85% of the substance of interest is bound, in a further embodimentmore than 95% of the substance of interest is bound to the stationaryphase.

In one embodiment the antibody is a therapeutic antibody. The term“therapeutic antibody” denotes an antibody which is tested in clinicalstudies for approval as human therapeutic and which can be administeredto an individual for the treatment of a disease. In another embodimentthe therapeutic antibody is a monoclonal antibody. In a furtherembodiment the therapeutic antibody is obtained from a great ape or ananimal transformed with a human antibody locus or a human monoclonalantibody or a humanized monoclonal antibody. In one embodiment thetherapeutic antibody is a human monoclonal antibody. In a furtherembodiment the therapeutic antibody is a humanized monoclonal antibody.Therapeutic antibodies are being used widely for the treatment ofvarious diseases such as oncological diseases (e.g. hematological andsolid malignancies including non-Hodgkin's lymphoma, breast cancer, andcolorectal cancer), immunological diseases, central nervous diseases,vascular diseases, or infectious diseases. Such antibodies are, in oneembodiment antibodies against ALK, adhesion related kinase receptor(e.g., Ax1), or ERBB receptors (e.g., EGFR, ERBB2, ERBB3, ERBB4), orerythropoietin-producing hepatocellular (EPH) receptors (e.g., EphA1;EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3,EphB4, EphB5, EphB6), or fibroblast growth factor (FGF) receptors (e.g.,FGFR1, FGFR2, FGFR3, FGFR4, FGFR5), or Fgr, or IGF-1R, or InsulinReceptor, or LTK, or M-CSFR, or MUSK, or platelet-derived growth factor(PDGF) receptors (e.g., PDGFR-A, PDGFR-B), or RET, or ROR1, or ROR2, orROS, or RYK, or vascular endothelial growth factor (VEGF) receptors(e.g., VEGF-R1/FLT1, VEGF-R2/FLK1, VEGF3), or tyrosine kinase withimmunoglobulin-like and EGF-like domains (TIE) receptors (e.g., TIE-1,TIE-2/TEK), or Tec, or TYRO10, or insulin-like growth factor (IGF)receptors (e.g., INS-R, IGF-IR, IR-R), or Discoidin Domain (DD)receptors (e.g., DDR1, DDR2), or receptor for c-Met (MET), or recepteurd'origine nantais (RON, also known as macrophage stimulating 1receptor), or FLT3 (fins-related tyrosine kinase 3), or colonystimulating factor 1 (CSF1) receptor, or receptor for c-kit (KIT, orSCF-R), or insulin receptor related (IRR) receptors, or CD19, or CD20,or HLA-DR, or CD33, or CD52, or G250, or GD3, or PSMA, or CD56, or CEA,or Lewis Y antigen, or IL-6 receptor.

The term “antibody” encompasses the various forms of antibody structuresincluding whole antibodies. The antibody is in one embodiment a humanantibody, a humanized antibody, a chimeric antibody, or a T cell antigendepleted antibody. Genetic engineering of antibodies is e.g. describedin Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984)6851-6855; U.S. Pat. No. 5,202,238; U.S. Pat. No. 5,204,244; Riechmann,L., et al., Nature 332 (1988) 323-327; Neuberger, M. S., et al., Nature314 (1985) 268-270; Lonberg, N., Nat. Biotechnol. 23 (2005) 1117-1125.Depending on the amino acid sequence of the constant region of the heavychains, antibodies are divided in the classes: IgA, IgD, IgE, and IgG.Some of these classes are further divided into subclasses (isotypes),i.e. IgG in IgG1, IgG2, IgG3, and IgG4, or IgA in IgA1 and IgA2.According to the class to which an antibody belongs are the heavy chainconstant regions of antibodies denoted as α (IgA), δ (IgD), ε (IgE), andγ (IgG), respectively. The term “antibody of human IgG1 class” forexample denotes an antibody in which the amino acid sequence of theconstant domains is derived from the amino acid sequence of human IgG1.The term includes human antibodies, humanized antibodies, chimericantibodies, and antibody conjugates.

The term “complete antibody” denotes an antibody which comprises twolight chain polypeptides (light chains) and two heavy chain polypeptides(heavy chains). Each of the heavy and light chain polypeptides containsa variable domain (variable region, generally the amino terminalportion) comprising binding regions that are able to interact with anantigen. Each of the heavy and light chain polypeptides comprises aconstant region (generally the carboxyl terminal portion). The variabledomain of a light or heavy chain in turn comprises different segments,i.e. four framework regions (FR) and three hypervariable regions (CDR).

The term “antibody conjugate” denotes a polypeptide comprising at leastone domain of an antibody heavy or light chain conjugated via a peptidebond to a further polypeptide. The further polypeptide can be anon-antibody peptide, such as a hormone, or toxin, or growth receptor,or antifusogenic peptide, or complement factor, or the like.

For the purification of recombinantly produced antibodies a combinationof different column chromatography steps can be employed. Generally aprotein A affinity chromatography is followed by one or two additionalseparation steps. The final purification step is a so called “polishingstep” for the removal of trace impurities and contaminants likeaggregated antibodies, residual HCP (host cell protein), DNA (host cellnucleic acid), viruses, or endotoxins.

The term “antibody in monomeric form” denotes an antibody molecule thatis not associated with a second antibody molecule, i.e. which is neithercovalently nor non-covalently bound to another antibody molecule. Theterm “antibody in aggregated form” denotes an antibody molecule which isassociated, either covalently or non-covalently, with at least oneadditional antibody molecule, and which is eluted in a single peak froma size exclusion chromatography column. The term “in monomeric form” asused herein not necessarily denotes that 100% of an antibody molecule ispresent in monomeric form. It denotes that an antibody is essentially inmonomeric form, i.e. at least 90% of the antibody is in monomeric from,in one embodiment at least 95% of the antibody is in monomeric form, inanother embodiment at least 99% of the antibody is in monomeric form, ina further embodiment at least 99.5% of the antibody is in monomericform, and in also an embodiment more than 99.8% of the antibody is inmonomeric form determined as peak area of a size exclusion chromatogram.The term “high molecular weight (HMW) form” denotes polymeric, i.e.aggregated, antibody, whereby this aggregate is still soluble in anaqueous buffered solution.

The term “100%” as used herein denotes that the amount of componentsother than a specified component is below the detection limit of thereferred to analytical method under the specified conditions.

The terms “90%”, “95%”, “99%”, “99.5%”, and “99.8%” as used within thisapplication denote no exact values but values within the accuracy of thereferred to analytical method under the specified conditions.

The term “elution compound” denotes a salt used for recovering of abound polypeptide from an ion exchange material, whereby the compoundincreases the conductivity of the buffer/solution. This can beaccomplished either by an increased buffer salt concentration or by theaddition of other salts, so called elution salts, to the bufferedsolution. Preferred elution salts are sodium citrate, sodium chloride,sodium sulphate, sodium phosphate, potassium chloride, potassiumsulfate, potassium phosphate, as well as other salts of citric andphosphoric acid, and any mixture of these components. In one embodimentthe elution compound is selected from sodium citrate, sodium chloride,potassium chloride, and mixtures thereof.

“Humanized” forms of non-human (e.g. rodent) antibodies are chimericantibodies that contain partial sequences derived from a non-humanantibody and from a human antibody. For the most part, humanizedantibodies are derived from a human antibody (recipient antibody), inwhich residues from a hypervariable region are replaced by residues froma hypervariable region of a non-human species (donor antibody), such asmouse, rat, rabbit, sheep, guinea pig, or non-human primate, having thedesired specificity and affinity. In some instances, framework region(FR) residues of the human antibody are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may comprisefurther modifications, e.g. amino acid residues that are not found inthe recipient antibody or in the donor antibody. Such modificationsresult in variants of such recipient or donor antibody, which arehomologous but not identical to the corresponding parent sequence. Thesemodifications can be made to further refine antibody performance.

In general, a humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human donor antibody and all or substantially all of the FRs arethose of a human recipient antibody. The humanized antibody optionallywill also comprise at least a portion of an antibody constant region,typically that of a human antibody.

In practice, humanized antibodies are typically human antibodies inwhich some hypervariable region residues and possibly some frameworkregion residues are substituted by residues from analogous sites inrodent or non-human primate antibodies.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies of the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which include different antibodies directedagainst different antigenic sites (determinants or epitopes), eachmonoclonal antibody is directed against a single antigenic site on anantigen. In addition to their specificity, monoclonal antibodies areadvantageous in that they may be synthesized uncontaminated by otherantibodies. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies and is not to be construed as requiring production of theantibody by any particular method.

The term “chimeric antibody” denotes an antibody comprising a variabledomain, i.e. binding region, from a first species and at least a portionof a constant region derived from a different second species.

Amino acid sequence variants of antibodies can be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody chains, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of residues within the amino acid sequences ofthe antibody. Any combination of deletion, insertion, and substitutioncan be made to arrive at the final construct, provided that the finalconstruct possesses the antigen binding properties as the parentantibody.

Conservative amino acid substitutions are shown in Table 1 under theheading of “preferred substitutions”. More substantial changes areprovided in Table 1 under the heading of “exemplary substitutions”, andas described below in reference to amino acid side chain classes. Aminoacid substitutions may be introduced into the antibody chains and theproducts screened for retention of the biological activity of the parentantibody.

TABLE Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “mobile phase” denotes any mixtures of water and/or aqueousbuffer and/or organic solvents being suitable to recover polypeptidesfrom a chromatography column. The term “to elute” or “eluting”,respectively, in the present context is used as known to the expertskilled in the art and denotes the dissolution, optionally thedisplacement, of adsorbed substance(s) from solids or adsorbents, whichare impregnated with fluids, i.e., the column material to which thesubstance(s) is/are adsorbed.

The term “adsorption” denotes the accumulation of substances from aliquid, e.g. a mobile phase, at the boundary phase formed between theliquid and a solid phase, wherein the latter is able to adsorb thesubstances of interest at its surface. This adsorption leads to anaccumulation of the adsorbed substances. The substance that is able toaccumulate the substance of interest at its surface is referred to asadsorbent and the adsorbed material as adsorbate. The term adsorption isusually distinguished from the term “absorption” which beyond theaccumulation at a surface also refers to the penetration of theaccumulated substances into the interior of the adsorbing solid orfluid. In general, adsorption is a physical process in which substancesusually molecules adhere to a surface of the adsorbent and thus, areaccumulated at the respective surface. The forces being responsible forthis adherence are considered to be physical forces rather than chemicalbonds and thus, adsorption is also known in the art as physicaladsorption or physisorption, which does not necessarily exclude chemicalbonding of substances to the surface. The physical forces involved inthe adsorption of substances to a surface are in most cases van derWaals-forces, London forces or dipole/dipole interactions, for examplehydrogen bonds, or dipole-induced dipole interactions, wherein theseterms are used as either explained herein or as normally used in contextwith adsorption.

In (column) chromatography usually solvents are used as eluent, i.e.,eluting agent in which the substance(s) which are to be eluted are atleast sufficiently soluble.

In one embodiment the non-ionic polymer is a hydrophilic non-ionicpolymer. In a further embodiment the non-ionic polymer is selected fromnon-ionic polyether, such as poly (ethylene glycol), poly (propyleneglycol) (PPG), PEG-PPG block copolymers, PEG-PPG copolymers, triblockcopolymers composed of poly (oxypropylene) (poly (propylene oxide)) andtwo poly (oxyethylene) (poly (ethylene oxide)) polymers (Poloxamer,Pluronic™), and other PEG-PPG-PEG triblock polymers.

The employed non-ionic polymer can be characterized besides itsmonomeric building block by its molecular weight given in Daltons (Da).The term “molecular weight” denotes with respect to polymers the meanmolecular weight of the polymer because polymeric compounds are notobtained with a defined molecular weight but in fact they have amolecular weight distribution. The molecular weight of a polymer isgiven in the form “about X Da”, whereby “X” denotes the mean value ofthe molecular weight of the polymer. The term “about” indicates that inthe polymer preparation, some molecules will have a weight more and somemolecules will have a weight less than the indicated mean molecularweight, i.e. the term about refers to a molecular weight distribution inwhich 95% of the polymer molecules have a molecular weight within +/−30%of the indicated molecular weight. For example, a molecular weight of3,500 Da denotes a range of from 2450 kDa to 4,550 Da. In one embodimentthe polymer molecules have a molecular weight within +/−20% of theindicated molecular weight. In one embodiment the polymer molecules havea molecular weight within +/−10% of the indicated molecular weight.

The non-ionic polymer can be present in a buffered solution, such as asalt containing buffered solution. The solution comprising thepolypeptide of interest can be obtained directly from the cultivationsupernatant of a cell which comprises a nucleic acid encoding thepolypeptide. The supernatant can be a clarified or a non-clarifiedcultivation supernatant.

In one embodiment one or more non-ionic polymers are present in thesolution applied to the chromatography material for recovering thepolypeptide, especially the antibody. In one embodiment the solutionapplied to the chromatography material for recovering the antibody inmonomeric from comprises one, or two, or three, or four, or fivedifferent non-ionic polymers. If more than one non-ionic polymer ispresent in the solution the sum of the concentrations of all non-ionicpolymers present in the solution is preferably within the range as givenherein.

In one embodiment one or more additives are present in the solutionapplied to the chromatography material for recovering the polypeptide,especially the antibody. In one embodiment the solution applied to thechromatography material for recovering the antibody in monomeric fromcomprises one, or two, or three, or four, or five different additives.If more than one additive is present in the solution the sum of theconcentrations of all additives present in the solution is preferablywithin the range as given herein.

Alternatively, the solution comprising the polypeptide of interest canbe the eluate from a preceding chromatography step.

The interactions between the polypeptide of interest and the ionexchange chromatography material can be adsorption, i.e. binding of thepolypeptide, or a retardation of the polypeptide in relation to othercomponent(s) also present in the solution.

In order to recover a polypeptide in monomeric form from the ionexchange chromatography material the adsorption is followed by a step ofeluting the adsorbed polypeptide of interest in monomeric form from theion exchange chromatography material. Elution of adsorbed polypeptidecan be effected by change of salt conditions, i.e. conductivity, and/orthe pH value as compared to the adsorption step.

With the exception of the addition of a non-ionic polymer and anadditive to the mobile phases during the recovering or eluting of thepolypeptide of interest from the ion exchange chromatography material,the chromatography steps of the method as reported herein are performedin accordance with conventional general operating conditions and wellknown principles in the field. Conventional chromatography columns areconveniently used, the size of which are adapted for each case, as isthe starting materials, buffers, matrices, including the functionalgroups of the ligands, etc.

The charge of a polypeptide may be changed by changing the pH value ofthe surrounding solution. It is well known, that an ion exchangematerial may be a strong ion exchange material, which means that it ischarged at all pH values, or a weak ion exchange material, which meansthat it is chargeable by shifting the pH.

Adding a non-ionic polymer and an additive to the mobile phase in an ionexchange chromatography recovering step as reported herein wasunexpectedly shown to increase the yield of the polypeptide in monomericform, as compared to conventional ion exchange chromatography. In oneembodiment the ion exchange chromatography is a cation exchangechromatography. In one embodiment the concentration of the non-ionicpolymer is at least about 5% by weight and the concentration of theadditive is at least 3% by weight.

There are in general no molecular weight exclusions for the non-ionicpolymer used in the method as reported herein. The molecular weight ofthe non-ionic polymer is chosen in a way that is as low as possiblewhile resulting in combination with the additive in an increased yieldof the polypeptide in monomeric form compared to the used of thenon-ionic polymer alone. The person skilled in the art knows that theaddition of any substance, i.e. of the non-ionic polymer and theadditive, affects (increases) the viscosity of the resulting solution.Therefore a person skilled in the art will appreciate that due to thephysicochemical properties of the non-ionic polymer and the additive anupper limit for the addition of these compounds exists.

The non-ionic polymer used as in the method as reported herein comprisesgroups that are rich in oxygen atoms. In one embodiment the non-ionicpolymer is an aqueous-soluble not-charged linear or branched polymer.Such non-ionic polymers are in one embodiment polyether, such as poly(ethylene glycol) (PEG), poly (propylene glycol) (PPG), PEG-PPG blockcopolymers, PEG-PPG copolymers, triblock copolymers composed of acentral hydrophobic chain of poly (oxypropylene) (poly(propylene oxide))flanked by two hydrophilic chains of poly (oxyethylene) (poly(ethyleneoxide)) (Poloxamer, Pluronic™), and other PEG-PPG-PEG triblock polymers,ethylhydroxyethylcellulose (EHEC) and similar polymers, polymerizedallylglycidyl ether, polymerized phenyl glycidyl ether, dextran, starch,cellulose, poly vinylpyrrolidone and various surfactants and othercompounds comprising these building blocks. In one embodiment thenon-ionic polymer is poly (ethylene glycol) (PEG). The term poly(ethylene glycol) also comprises poly (ethylene glycol)s which have beenmodified.

Any molecular weight non-ionic polymer can be used in the method asreported herein. However, the optimal concentration and ratio of thenon-ionic polymer and the additive may vary for every protein and, inmost cases, there is a preferable molecular weight of the non-ionicpolymer and a preferable ratio of the non-ionic polymer to the additive.

In one embodiment the non-ionic polymer is poly (ethylene glycol).

Poly (ethylene glycol) can be used as a general model non-ionicpolypeptide for behavior of non-ionic polymers within the method asreported herein. Thus, while poly (ethylene glycol) is used as anexample herein it should be kept in mind that the provided informationalso applies to other non-ionic polymers, including to thosespecifically listed herein above.

Poly (ethylene glycol) is available from a number of commercial sources.In one embodiment the poly (ethylene glycol) has a molecular weight inthe range of from about 100 Da to about 40,000 Da. In one embodiment thepoly (ethylene glycol) has a molecular weight between about 400 Da andabout 10,000 Da. In one embodiment the poly (ethylene glycol) has amolecular weight between about 1,000 Da and about 8,000 Da. In otherembodiments, a mixture of different sized poly (ethylene glycol)s isutilized.

In one embodiment the poly (ethylene glycol) has a molecular weightbetween about 2,450 Da and about 4,550 Da. In one embodiment the poly(ethylene glycol) has a molecular weight between about 2,625 Da andabout 4,375 Da. In one embodiment the poly (ethylene glycol) has amolecular weight between about 2,800 Da and about 4,200 Da. In oneembodiment the poly (ethylene glycol) has a molecular weight betweenabout 3,150 Da and about 3,850 Da.

In one embodiment the poly (ethylene glycol) is a linear or branchedpoly (ethylene glycol).

Lower molecular weight poly (ethylene glycols) can require a higher massconcentration to be used in order to have a similar effect compared tohigher molecular weight poly (ethylene glycol)s.

Likewise lower concentrations of a poly (ethylene glycol) of a specifiedmolecular weight can be employed to produce larger polypeptide inmonomeric form, such as antibodies and fusion proteins, compared tothose concentrations required to effect the same purity of smallerpolypeptides. For example, an antibody of the class IgA, which is mostlypresent as a dimer, having a molecular weight of about 320 kDa willrequire a lower concentration of poly (ethylene glycol) compared to anantibody of the class IgG having a molecular weight of about 150 kDa.The retention of aggregates, complexes, and other large molecules cangenerally be increased more compared to that of the monomericpolypeptide. Also can a lower concentration of poly (ethylene glycol) berequired to increase the retention of polypeptides that interact/areadsorbed strongly to the cation exchange chromatography materialcompared to the required concentration of poly (ethylene glycol)required to increase the adsorption of polypeptides that are normallyadsorbed weakly to the cation exchange chromatography material.

The additive is a substance that can reduce or even preventprecipitation that is induced by the presence of the non-ionic polymer.

In one embodiment the additive has a conductivity that is sufficientlylow to not interfere with a cation exchange chromatography. In anotherembodiment the additive has no detectable buffering capacity.

In one embodiment the additive is a zwitterion, or an amino acid (suchas glycine, bicine, tricine, alanine, proline, Betaine), or urea, or anurea derivative (such as alkyl ureas (methyl urea, ethyl urea, etc.)),or alkylene glycols (such as ethylene glycol or propylene glycol), or anampholyte (such as amino-sulfonic acid based buffers MES, MOPS, HEPES,PIPES and CAPS buffer), or CHAPSO, or a natural product (such as certainalkaloids and Betaines), or a sugar (such as glucose, sucrose,raffinose), or a polyol (such as glycerol, or xylitol, or sorbitol).

In one embodiment the additive is a zwitterion.

In one embodiment the additive is an amino acid. In a further embodimentthe amino acid is glycine, or bicine, or tricine, or alanine, orproline, or Betaine.

In one embodiment the additive is urea or a urea derivative. In afurther embodiment the additive is methyl urea or ethyl urea.

In one embodiment the additive is an alkylene glycol. In a furtherembodiment the alkylene glycol is ethylene glycol or propylene glycol.

In one embodiment the additive is an ampholyte. In a further embodimentthe ampholyte is MES, or MOPS, or HEPES, or PIPES, or CAPS.

In one embodiment the additive is a natural product. In a furtherembodiment the natural product is an alkaloid or a Betaine.

In one embodiment the additive is a sugar. In a further embodiment thesugar is glucose, or sucrose, or raffinose, or fructose.

In one embodiment the additive is a polyol. In a further embodiment thepolyol is glycerol, or xylitol, or sorbitol.

For any additive it has to be kept in mind that the individualsolubility limit has to be considered when determining the respectiveamount or concentration employed.

If an amino acid is employed as additive it has to be kept in mind thata charged side chain will require the use of additional acid or base inorder to adjust the pH value of the solution. By the addition of surplusacid or base the ionic strength of the mobile phase is change which inturn might have an influence on the chromatography result.

For example, glycine can be used as additive in the method as reportedherein. Glycine is a zwitterionic compound, more precisely glycine is anamino acid. At pH values generally employed in cation exchangechromatography methods glycine does not contributes to overallconductivity and, thus, should not interfere in the cation exchangechromatography method. Additionally, glycine does not exhibit abuffering effect in aqueous solution it will also not effect the pHvalue of the mobile phase. Glycine can be used as additive in the methodas reported herein in a concentration of from about 50 mM to 5 M. In oneembodiment glycine has a concentration of from about 100 mM to about 4M. In another embodiment glycine has a concentration of from about 250mM to about 3 M. In a further embodiment glycine has a concentration offrom about 500 mM to about 2 M. In still another embodiment glycine hasa concentration of about 1 M. If more than one additive is used theconcentrations of the individual additives can be lower than thoseexemplarily given in the paragraph above.

It has to be pointed out that any additive can be used at aconcentration that is higher than the concentration necessary to achievethe intended effect. Especially a person skilled in the art candetermine the additive concentration range in that the effect is presentand that can be tolerated in the method as reported herein.

In one embodiment when the additive is urea or a urea derivative theadditive is present in a concentration up to 6 molar. In one embodimentthe additive is present in a concentration below 2 molar.

As outlined above the concentration of the non-ionic polymer and theadditive in the recovering solution can vary. Generally, anyconcentration of poly (ethylene glycol) can be used. In one embodimentconcentration of poly (ethylene glycol) is at least about 0.5% byweight. In one embodiment the concentration of poly (ethylene glycol) isat least about 0.5% by weight and at most about 20% by weight. In afurther embodiment the concentration of poly (ethylene glycol) is atleast about 5% by weight and at most about 15% by weight. In anotherembodiment the concentration of poly (ethylene glycol) is at least about7% by weight and at most about 13% by weight.

The concentration of the non-ionic polymer can be held constant or itcan be changed during the course of the chromatography. This changeincludes but is not limited to a gradient of increasing or decreasingconcentration, or with step-wise changes in concentration.

In one embodiment the non-ionic polymer has a concentration of fromabout 0.5% to 20% by weight and/or the additive has a concentration offrom 0.5% to 40% by weight.

In one embodiment the non-ionic polymer has a concentration of fromabout 5% to 15% by weight and/or the additive has a concentration offrom 0.5% to 40% by weight.

In one embodiment the non-ionic polymer has a concentration of fromabout 5% to 15% by weight and/or the additive has a concentration offrom 3% to 25% by weight.

In one embodiment the non-ionic polymer has a concentration of fromabout 7% to 13% by weight and/or the additive has a concentration offrom 3% to 25% by weight.

In one embodiment the non-ionic polymer has a concentration of fromabout 7% to 13% by weight and/or the additive has a concentration offrom 12% to 25% by weight.

In one embodiment the non-ionic polymer has a concentration of fromabout 7% to 13% by weight and/or the additive has a concentration offrom 12% to 20% by weight.

The use or addition of a non-ionic polymer results in a reduction of thesolubility of a polypeptide, especially of an antibody, concomitantlypresent in this solution. In the following Table the dependency of thesolubility of an anti-IL17 antibody (5 mg/ml) as an exemplarypolypeptide at room temperature (RT) and at 4° C. on the concentrationof added poly (ethylene glycol) 3,500 Da is shown (50 mM sodiummalonate, 90 mM NaCl, pH 6.5).

TABLE RT: RT: 4° C.: 4° C.: protein protein protein protein conc. conc.conc. conc. PEG- (UV280) [fraction of (UV280) [fraction of concentration[mg/ml] initial conc.] [mg/ml] initial conc.]   0% 5.0 100% 5.1 100%  1% 5.1 100% 5.1 100% 2.5% 5.1 100% 5.1 100% 5.0% 5.1 100% 5.8 100%7.5% 5.0 100% 2.1 42%  10% 2.5 50% 0.5 10%

In the following Table the dependency of the solubility of an anti-IL17antibody (5 mg/ml) as an exemplary polypeptide at room temperature andat 4° C. on the concentration of added sodium chloride is shown (50 mMsodium malonate, 7.5 wt-% poly(ethylene glycol) with MW of 3,500 Da, pH6.5).

TABLE RT: 4° C.: RT: protein 4° C.: protein protein conc. protein conc.conc. [fraction conc. [fraction NaCl- (UV280) of initial (UV280) ofinitial concentration [mg/ml] conc.] [mg/ml] conc.]  0 mM 2.9 61% 0.714%  45 mM 3.3 67% 0.8 17%  90 mM 3.6 75% 0.9 19% 135 mM 4.0 83% 1.0 20%180 mM 4.6 95% 1.6 33%

In one embodiment the non-ionic polymer is poly (ethylene glycol) andthe additive is a polyol. In FIG. 3 the solubility of an antibody in asolution comprising poly (ethylene glycol) of a molecular weight ofabout 3,500 Da at a concentration of 10% by weight as an example of anon-ionic polymer and different polyols at varying concentration asadditive are shown.

In one embodiment the non-ionic polymer is poly (ethylene glycol) with amolecular weight of about 3,500 Da and the additive is an amino acid. InFIG. 4 the solubility of an antibody in a solution comprising poly(ethylene glycol) with a molecular weight of about 3,500 Da at aconcentration of 10% by weight as an example of a non-ionic polymer anddifferent amino acids at varying concentration as additive is shown.

In one embodiment the non-ionic polymer is poly (ethylene glycol) with amolecular weight of about 3,500 Da and the additive is a sugar. In FIG.5 the solubility of an antibody in a solution comprising poly (ethyleneglycol) with a molecular weight of about 3,500 Da at a concentration of10% by weight as an example of a non-ionic polymer and different sugarsat varying concentration as additive is shown.

As can be seen from the following Table separations in the presence ofonly the additive do not provided for an improvement in the resolutionvalue.

TABLE anti-IL17 anti-CSF-1R resolution value antibody antibody None 0.460.64 Glycine 0.52 0.61 Glutamate 0.49 0.68 Arginine 0.53 0.73 Glycerin0.49 0.68 Urea 0.46 0.66 Mannitol 0.46 0.69 Sucrose 0.46 0.39 Trehalose0.45 0.60

In one embodiment the term “polyol” denotes a compound that has at leastthree hydroxyl groups, i.e. is a triol. Thus in one embodiment a polyolis selected from a triol that has three free hydroxyl groups, or atetrol that has four free hydroxyl groups, or a pentaol that has fivefree hydroxyl groups, or a hexaol that has six free hydroxyl groups, ora heptaol that has seven free hydroxyl groups, or an octaol that haseight free hydroxyl groups.

The reduction in solubility by the addition of a non-ionic polymer, e.g.poly (ethylene glycol), can be prevented by the addition of an additive,e.g. sorbitol.

Thus, one aspect as reported herein is a method for determining theconcentration of a non-ionic polymer and an additive for use in an ionexchange chromatography of a polypeptide comprising the following steps:

-   -   determining in solution in the absence of the additive the        concentration of the non-ionic polymer at which less than 50% of        the polypeptide remain in solution,    -   determining in solution in the presence of a concentration of        the non-ionic polymer, which has been determine in the previous        step, the concentration of the additive at which more than 95%        of the polypeptide remain in solution,        thereby determining the concentration of a non-ionic polymer and        an additive.

In the following Table the dependency of the solubility of an anti-IL17antibody (10 mg/ml) as an exemplary polypeptide at room temperature onthe concentration of added sorbitol as an example of an additive also atdifferent sodium chloride concentrations is shown (10 mM sodium citrate,poly(ethylene glycol) with a molecular weight of about 3,500 Da, pH5.0).

TABLE additive sodium protein conc. (sorbitol) chloride PEG 3,500c(UV280) [fraction of [wt-%] [mM] [wt-%] [mg/ml] initial conc.] 0 0 010.7 ~100% 0 0 5 10.6 ~100% 0 0 10 1.96  19.6% 0 250 0 10.3 ~100% 0 2505 10.6 ~100% 0 250 10 1.77  17.7% 0 500 0 10.5 ~100% 0 500 5 10.6 ~100%0 500 10 1.57  15.7% 7.5 0 0 11.1 ~100% 7.5 0 5 10.6 ~100% 7.5 0 10 5.26 52.6% 7.5 250 0 10.7 ~100% 7.5 250 5 10.6 ~100% 7.5 250 10 5.83  58.3%7.5 500 0 10.7 ~100% 7.5 500 5 10.6 ~100% 7.5 500 10 4.66  46.6% 15 0 010.7 ~100% 15 0 5 10.8 ~100% 15 0 10 11.0 ~100% 15 250 0 10.8 ~100% 15250 5 10.7 ~100% 15 250 10 10.4 ~100% 15 500 0 10.7 ~100% 15 500 5 10.6~100% 15 500 10 10.5 ~100%

In the following Table the dependency of the solubility of an anti-IL17antibody (10 mg/ml) and an anti-CSF-1R antibody (10 mg/ml) as anexemplary polypeptides at room temperature on the concentration of addedglycerol as an example of an additive is shown (10 mM sodium citrate,poly(ethylene glycol) with MW of 3,500 Da, pH 5.0).

TABLE protein conc. c(UV280) protein conc. c(UV280) anti-IL17 anti-anti-CSF-1R additive PEG- anti-IL17 antibody CSF-1R antibody (glycerol)3,500 antibody [fraction of antibody [fraction of [wt-%] [wt-%] [mg/ml]initial conc.] [mg/ml] initial conc.] 0 0 9.65 96.5% 9.82 98.2% 0 5 10.0 100% 10.0  100% 0 10 2.13 21.3% 0.59  5.9% 7.5 0 9.76 97.6% 9.96 99.6%7.5 5 9.88 98.8% 10.2 ~100%  7.5 10 3.28 32.8% 1.56 15.6% 15 0 9.8298.2% 9.88 98.8% 15 5 9.92 99.2% 10.2 ~100%  15 10 6.70 67.0% 3.04 30.4%20 0 9.95 99.5% 10.2 ~100%  20 5 9.89 98.9% 10.2 ~100%  20 10 8.46 84.6%5.54 55.4% 25 0 10.3 ~100%  9.87 98.7% 25 5 10.0  100% 9.86 98.6% 25 109.92 99.2% 8.94 89.4% 30 0 10.1 ~100%  9.77 97.7% 30 5 9.92 99.2% 10.1~100%  30 10 9.85 98.5% 10.0  100%

In the following Table the dependency of the solubility of an anti-IL17antibody (10 mg/ml) and an anti-CSF-1R antibody (10 mg/ml) as anexemplary polypeptides at room temperature on the concentration of addedxylitol as an example of an additive is shown (10 mM sodium citrate,poly(ethylene glycol) with MW of 3,500 Da, pH 5.0).

TABLE protein conc. protein conc. c(UV280) anti-IL17 c(UV280)anti-CSF-1R additive PEG- anti-IL17 antibody anti-CSF-1R antibody(xylitol) 3,500 antibody [fraction of antibody [fraction of [wt-%][wt-%] [mg/ml] initial conc.] [mg/ml] initial conc.] 15 0 10.1 ~100%10.2 ~100% 15 10 9.01  90.1% 5.15  51.5% 20 0 10.2 ~100% 10.1 ~100% 2010 10.3 ~100% 10.1 ~100%

In the following Table the dependency of the solubility of an anti-IL17antibody (10 mg/ml) and an anti-CSF-1R antibody (10 mg/ml) as anexemplary polypeptides at room temperature on the concentration of addedglycine as an example of an additive is shown (10 mM sodium citrate,poly(ethylene glycol) with MW of 3,500 Da, pH 5.0).

TABLE protein conc. c(UV280) protein conc. c(UV280) anti-IL17 anti-anti-CSF-1R additive PEG- anti-IL17 antibody CSF-1R antibody (glycine)3,500 antibody [fraction of antibody [fraction of [wt-%] [wt-%] [mg/ml]initial conc.] [mg/ml] initial conc.] 0 0 9.82 98.2% 9.94 99.4% 0 5 10.0 100% 10.0  100% 0 10 1.60 16.0% 0.53  5.3% 1 0 9.85 98.5% 9.72 97.2% 110 3.80 38.0% 1.49 14.9% 2 0 9.73 97.3% 9.83 98.3% 2 10 7.18 71.8% 3.1931.9% 4 0 9.84 98.4% 9.77 97.7% 4 5 9.96 99.6% 10.1 ~100%  4 10 10.5~100%  9.71 97.1% 8 0 9.89 98.9% 9.95 99.5% 8 5 9.87 98.7% 9.94 99.4% 810 9.90 99.0% 11.3 ~100% 

In the following Table the dependency of the solubility of an anti-IL17antibody (10 mg/ml) and an anti-CSF-1R antibody (10 mg/ml) as anexemplary polypeptides at room temperature on the concentration of addedproline as an example of an additive is shown (10 mM sodium citrate,poly(ethylene glycol) with MW of 3,500 Da, pH 5.0).

TABLE protein conc. c(UV280) protein conc. c(UV280) anti-IL17 anti-anti-CSF-1R additive PEG- anti-IL17 antibody CSF-1R antibody (proline)3,500 antibody [fraction of antibody [fraction of [wt-%] [wt-%] [mg/ml]initial conc.] [mg/ml] initial conc.] 1 0 9.74 97.4% 9.75 97.5% 1 103.86 38.6% 1.63 16.3% 2 0 9.78 97.8% 9.99 99.9% 2 10 8.38 83.8% 3.6136.1% 4 0 9.93 99.3% 10.0  100% 4 10 9.85 98.5% 10.1 >100%  8 0 9.9899.8% 10.1 >100%  8 10 10.1 >100%  10.2 >100% 

In the following Table the dependency of the solubility of an anti-IL17antibody (10 mg/ml) and an anti-CSF-1R antibody (10 mg/ml) as anexemplary polypeptides at room temperature on the concentration of addedBetaine as an example of an additive is shown (10 mM sodium citrate,poly(ethylene glycol) with MW of 3,500 Da, pH 5.0).

TABLE protein conc. c(UV280) protein conc. c(UV280) anti-IL17 anti-anti-CSF-1R additive PEG- anti-IL17 antibody CSF-1R antibody (Betaine)3,500 antibody [fraction of antibody [fraction of [wt-%] [wt-%] [mg/ml]initial conc.] [mg/ml] initial conc.] 0 0 9.66 96.6% 9.82 98.2% 0 5 9.8198.1% 9.97 99.7% 0 10 1.53 15.3% 0.43  4.3% 1 0 9.57 95.7% 9.83 98.3% 110 7.11 71.1% 2.28 22.8% 2.5 0 9.60 96.0% 9.60 96.0% 2.5 10 9.68 96.8%9.89 98.9% 5 0 9.73 97.3% 9.81 98.1% 5 10 9.57 95.7% 10.0 100% 7.5 09.65 96.5% 9.86 98.6% 7.5 5 9.98 99.8% 9.99 99.9% 7.5 10 10.5 ~100% 10.2 ~100%  15 0 9.75 97.5% 9.87 98.7% 15 5 10.1 ~100%  10.1 ~100%  1510 10.3 ~100%  10.7 ~100% 

In the following Table the dependency of the solubility of an anti-IL17antibody (10 mg/ml) and an anti-CSF-1R antibody (10 mg/ml) as anexemplary polypeptides at room temperature on the concentration of addedsucrose as an example of an additive is shown (10 mM sodium citrate,poly(ethylene glycol) with MW of 3,500 Da, pH 5.0).

TABLE protein conc. c(UV280) protein conc. c(UV280) anti-IL17 anti-anti-CSF-1R additive PEG- anti-IL17 antibody CSF-1R antibody (sucrose)3,500 antibody [fraction of antibody [fraction of [wt-%] [wt-%] [mg/ml]initial conc.] [mg/ml] initial conc.] 0 0 9.64 96.4% 9.74 97.4% 0 5 10.0 100% 10.0  100% 0 10 1.63 16.3% 0.66  6.6% 7.5 0 9.56 95.6% 9.76 97.6%7.5 5 10.2 ~100%  10.3 ~100%  7.5 10 4.49 44.9% 1.58 15.8% 15 0 9.8298.2% 9.90 99.0% 15 5 10.4 ~100%  10.4 ~100%  15 10 7.80 78.0% 3.3333.3% 20 0 9.71 97.1% 9.80 98.0% 20 10 9.96 99.6% 10.2 ~100%  25 0 9.6596.5% 10.0  100% 25 10 9.73 97.3% 9.95 99.5%

In the following Table the dependency of the solubility of an anti-IL17antibody (10 mg/ml) and an anti-CSF-1R antibody (10 mg/ml) as anexemplary polypeptides at room temperature on the concentration of addedfructose as an example of an additive is shown (10 mM sodium citrate,poly(ethylene glycol) with MW of 3,500 Da, pH 5.0).

TABLE protein conc. c(UV280) protein conc. c(UV280) anti-IL17 anti-anti-CSF-1R additive PEG- anti-IL17 antibody CSF-1R antibody (fructose)3,500 antibody [fraction of antibody [fraction of [wt-%] [wt-%] [mg/ml]initial conc.] [mg/ml] initial conc.] 0 0 9.79 97.9% 9.56 95.6% 0 5 9.9399.3% 9.85 98.5% 0 10 0.98  9.8% 0.42  4.2% 7.5 0 9.76 97.6% 9.78 97.8%7.5 5 9.97 99.7% 10.1 >100%  7.5 10 3.85 38.5% 1.42 14.2% 15 0 10.0 100% 9.49 94.9% 15 5 10.1 >100%  10.0  100% 15 10 8.23 82.3% 3.76 37.6%20 0 9.65 96.5% 9.91 99.1% 20 10 9.92 99.2% 10.3 >100%  25 0 10.0  100%9.61 96.1% 25 10 9.99 99.9% 10.3 >100% 

In the following Table the dependency of the solubility of an anti-IL17antibody (10 mg/ml) and an anti-CSF-1R antibody (10 mg/ml) as anexemplary polypeptides at room temperature on the concentration of addedglucose as an example of an additive is shown (10 mM sodium citrate,poly(ethylene glycol) with MW of 3,500 Da, pH 5.0).

TABLE protein conc. c(UV280) protein conc. c(UV280) anti-IL17 anti-anti-CSF-1R additive PEG- anti-IL17 antibody CSF-1R antibody (glucose)3,500 antibody [fraction of antibody [fraction of [wt-%] [wt-%] [mg/ml]initial conc.] [mg/ml] initial conc.] 7.5 0 9.84 98.4% 97.8 97.8% 7.5 105.35 53.5% 2.39 23.9% 15 0 9.98 99.8% 9.92 99.2% 15 10 9.63 96.3% 5.8458.4% 20 0 10.1 ~100%  10.1 ~100%  20 10 10.0  100% 8.08 80.8% 25 0 10.5~100%  10.4 ~100%  25 10 10.4 ~100%  10.3 ~100% 

When a non-ionic polymer is combined with an additive in a method asreported herein an improved resolution value can be achieved. In thefollowing Table the obtained resolution value of a combination of poly(ethylene glycol) 3,500 at a concentration of 10% by weight as anexample of a non-ionic polymer and an additive at the listedconcentration in a chromatographic separation of an anti-IL17 antibodyis shown.

TABLE anti- anti- IL17 CSF-1R resolution value antibody antibody 5% PEG,3,500 Da 0.75 0.66 MW D-sorbitol, 15 wt-% 0.85 0.86 glycine, 8 wt-% 0.850.80 L-proline, 8 wt-% 0.83 0.97 Betaine, 8 wt-% 0.86 0.86 saccharose,25 wt-% 0.73 0.84 D-fructose, 25 wt-% 0.79 0.91 D-glucose, 25 wt-% 0.810.97 xylitol, 20 wt-% 0.76 0.94 glycerol, 30 wt-% 0.77 0.90

If 10% by weight of poly (ethylene glycol) is used without an additivethe antibody precipitates. Therefore, the reference value in the abovetable was determined in the presence of 5% by weight of the non-ionicpolymer.

In the following Table the change in the host cell protein content andthe host cell DNA content of a sample subjected to a method as reportedherein is shown. It has to be pointed out that in order to determine ofthe host cell protein content and the host cell DNA content the fractionsize had to be increased which resulted in a reduced resolution value.The elution is effected in all presented examples by increasing thesodium chloride concentration in the mobile phase.

TABLE HCP host cell DNA resolution reduction by reduction by elutionconditions value [%] [%] anti-IL17 antibody no non-ionic polymer, 0.4485.5 61.8 no additive  5 wt-% PEG-3,500 0.70 94.5 increase 10 wt-%PEG-3,500, 0.78 98.4 increase 15 wt-% D-sorbitol anti-CSF-1R antibody nonon-ionic polymer, 0.56 98.6 80.9 no additive  5 wt-% PEG-3,500 0.6699.5 77.7 10 wt-% PEG-3,500, 0.88 99.7 17.0 15 wt-% D-sorbitol

As can be seen from the table above in a chromatographic methodcomprising a non-ionic polymer and an additive in the recoveringsolution the HCP (host cell protein) content in the recovered antibodypreparation can be reduced compared to methods performed in the absenceof this combination.

Thus, one aspect as reported herein is a method for producing anantibody preparation with reduced host cell protein content whereby thepreparation is obtained from a mammalian cell cultivation supernatant,especially a CHO cell cultivation supernatant, comprising the followingsteps:

-   -   applying a solution comprising a non-ionic polymer and an        additive to a chromatography material to which an antibody had        been adsorbed, whereby the antibody in monomeric form remains        adsorbed to an ion exchange chromatography material, and    -   recovering the antibody in monomeric form from the ion exchange        chromatography material by applying a solution comprising a        non-ionic polymer, an additive, and an elution compound, and        thereby producing the antibody preparation with reduced host        cell protein content.

Another aspect as reported herein is a method for producing an antibodycomprising the following steps:

-   -   a) cultivating a mammalian cell comprising a nucleic acid        encoding the antibody and recovering the antibody from the cell        or the cultivation medium,    -   b) purifying the antibody with a cation exchange chromatography        method comprising the following steps:        -   applying a solution comprising a non-ionic polymer and an            additive to a chromatography material to which an antibody            had been adsorbed, whereby the antibody in monomeric form            remains adsorbed to an ion exchange chromatography material,            and        -   recovering the antibody in monomeric form from the ion            exchange chromatography material by applying a solution            comprising a non-ionic polymer, an additive, and an elution            compound, and thereby producing the antibody.

In the following Table the resolution values obtained with differentcation exchange chromatography materials with an anti-IL17 antibody areshown. The elution is effected in all presented examples by increasingthe sodium chloride concentration in the mobile phase.

TABLE chromatography resolution elution conditions material value nonon-ionic polymer, SP Sepharose FF 0.29 no additive no non-ionicpolymer, Toyopearl CM-650M 0.53 no additive no non-ionic polymer, CMSepharose FF 0.29 no additive 5 wt-% PEG-3,500 SP Sepharose FF 0.45 5wt-% PEG-3,500 Toyopearl CM-650M 0.67 5 wt-% PEG-3,500 CM Sepharose FF0.44 10 wt-% PEG-3,500 SP Sepharose FF 0.51 15 wt-% D-sorbitol 10 wt-%PEG-3,500 Toyopearl CM-650M 0.76 15 wt-% D-sorbitol 10 wt-% PEG-3,500 CMSepharose FF 0.51 15 wt-% D-sorbitol

In the following Table the resolution values obtained with a method asreported herein using poly (ethylene glycol) 400 Da MW and 10,000 Da MWas non-ionic polymer with different molecular weights and the polyolsorbitol as additive are shown with an anti-IL17 antibody.

TABLE resolution elution conditions value 10 wt-% PEG-400; 15 wt-%D-sorbitol 0.52 10 wt-% PEG-400; 15% D-sorbitol 0.56 10 wt-% PEG-400;15% D-sorbitol 0.57 average: 0.55 10 wt-% PEG-10.000; 15% D-sorbitol0.58 10 wt-% PEG-10.000; 15% D-sorbitol 0.70 10 wt-% PEG-10.000; 15%D-sorbitol 0.65 average: 0.64

Depending on the envisaged purity after performing a method as reportedherein the recovery of the monomeric polypeptide varies depending on thecombination of the concentration of the non-ionic polymer and theadditive.

In the following this interrelation is exemplified with poly (ethyleneglycol) as non-ionic polymer, sorbitol as additive, and an anti-IL17antibody as polypeptide.

In the following Table the recovery and the final purity of monomericanti-IL17 antibody with a purity of at least 99% in the pooled(combined) elution fractions depending on the concentration of poly(ethylene glycol) and sorbitol is shown.

TABLE sorbitol [wt-%] 0 3 5 10 15 20 30 40 recovery monomer [%] PEG- 082.3 77.9 3,500 5 87.0 84.5 [wt-%] 6 89.0 8 82.7 9 86.1 92.6 10 86.188.5 89.2 88.6 90.0 84.9 88.1 15 87.1 purity monomer [%] PEG- 0 99.599.5 3,500 5 99.3 99.3 [wt-%] 6 99.3 8 98.9 9 99.0 99.1 10 99.1 98.898.9 99.3 99.2 99.1 99.0 15 99.2

The values for the empty cells were not determined.

In the following Table the recovery and the final purity of monomericanti-IL17 antibody with a purity of at least 99.5% in the pooled(combined) elution fractions depending on the concentration of poly(ethylene glycol) and sorbitol is shown.

TABLE sorbitol [wt-%] 0 3 5 10 15 20 30 40 recovery monomer [%] PEG- 082.3 77.9 3,500 5 81.3 78.7 [wt-%] 6 83.3 8 81.1 9 84.3 92.0 10 84.985.3 88.2 87.1 87.0 81.5 82.8 15 86.0 purity monomer [%] PEG- 0 99.599.5 3,500 5 99.8 99.9 [wt-%] 6 99.8 8 99.8 9 99.5 99.5 10 99.5 100.099.5 99.7 99.7 99.8 99.6 15 99.5

The values for the empty cells were not determined.

In the following Table the recovery and the final purity of monomericanti-IL17 antibody with a purity of at least 99.9% in the pooled(combined) elution fractions depending on the concentration of poly(ethylene glycol) and sorbitol is shown.

TABLE sorbitol [wt-%] 0 3 5 10 15 20 30 40 recovery monomer [%] PEG- 066.3 63.9 3,500 5 67.9 78.7 [wt-%] 6 66.0 8 75.1 9 76.9 89.1 10 80.485.3 83.1 84.5 81.3 76.0 64.6 15 80.9 purity monomer [%] PEG- 0 99.9100.0 3,500 5 100.0 99.9 [wt-%] 6 100.0 8 100.0 9 99.9 99.9 10 99.9100.0 100.0 99.9 99.9 100.0 100.0 15 99.9

The values for the empty cells were not determined.

In the following Table the recovery and the final purity of monomericanti-IL17 antibody with a purity of 100% in the pooled (combined)elution fractions depending on the concentration of poly (ethyleneglycol) and sorbitol is shown.

TABLE sorbitol [wt-%] 0 3 5 10 15 20 30 40 recovery monomer [%] PEG- 041.1 63.9 3,500 5 67.9 67.1 [wt-%] 6 66.0 8 75.1 9 67.0 84.7 10 75.885.3 83.1 68.7 70.5 76.0 64.6 15 75.5 purity monomer [%] PEG- 0 100.0100.0 3,500 5 100.0 100.0 [wt-%] 6 100.0 8 100.0 9 100.0 100.0 10 100.0100.0 100.0 100.0 100.0 100.0 100.0 15 100.0

The values for the empty cells were not determined.

Generally a chromatographic separation step comprises several sub-steps:

-   -   equilibrating the packed chromatographic material in a        chromatography column,    -   applying the solution comprising the polypeptide of interest to        the equilibrated chromatography material under conditions        suitable for the adsorption/binding of the polypeptide of        interest to the chromatography material and thereby loading the        chromatography column,    -   optionally washing the chromatographic material without        desorbing/eluting the polypeptide of interest from the        chromatographic material,    -   recovering the polypeptide of interest by applying a recovering        solution to the chromatographic material and thereby        desorbing/eluting the polypeptide of interest from the        chromatographic material.

In one embodiment the cation exchange chromatography material in thecolumn is equilibrated prior to the applying of the solution comprisingthe polypeptide to the column. This can be accomplished by applying anequilibration buffer to the column to establish a suitable pH value,and/or conductivity, and/or concentration of salt inside or on thecation exchange chromatography material.

In one embodiment the solution comprising the polypeptide is adjusted toconditions comparable to those to which the cation exchangechromatography column is equilibrated.

Different methods for loading of the chromatography column with thepolypeptide of interest are possible. In one embodiment the polypeptideof interest is added to the solution and is loaded onto the column. Inanother embodiment the polypeptide of interest is added after the columnis equilibrated and/or washed. In one embodiment the equilibratingcomprises the applying of a binding buffer before loading of thesolution onto the chromatographic material.

As outlined above is the method as reported herein performed inbind-and-elute mode.

The recovery of the polypeptide in monomeric form in the method asreported herein can be carried out using a linear gradient, a stepgradient, or a combination of a linear and a step gradient. A lineargradient can be, for example, used to further improve the resolution ofthe polypeptide in monomeric form from the polypeptide in aggregatedform and/or from other contaminants such as host cell protein. A stepgradient can be used in order to reduce the volume of recoveredpolypeptide solution. Selection and use of any gradient in the recoveryof the polypeptide from the cation exchange chromatography material caneasily be made by a person skilled in the art.

In one embodiment the application of the solution comprising thepolypeptide is followed by the application of a buffered wash solutionto the chromatography material, often of the same composition as theequilibration buffer.

In one embodiment the concentration of the non-ionic polymer is heldconstant during elution while the pH value is changed and/or theconcentration of the salt causing elution is increased.

In one embodiment the concentration of the non-ionic polymer isincreased during elution while the pH value is changed and/or theconcentration of a salt causing elution is increased.

In one embodiment the concentration of the non-ionic polymer isdecreased during elution while the pH value and/or the concentration ofthe salt are held constant.

After use, the cation exchange chromatography material can be cleaned,sanitized, and stored in an appropriate agent.

The method as reported herein can practiced in combination with one ormore other chromatographic steps or methods, such as but not limited toprotein A affinity chromatography and other forms of affinitychromatography, hydrophobic interaction chromatography, or other mixedmode chromatography.

The method as reported herein can be practiced in a packed bed column, afluidized/expanded bed column containing the cation exchangechromatography material, and/or a batch operation where the cationexchange chromatography material is mixed with the polypeptidepreparation for a certain time.

The method as reported herein is useful to produce biologically activepolypeptides in monomeric form. Such biologically active polypeptidesare in one embodiment selected from antibodies, antibody variant,antibody conjugates. In another embodiment the polypeptide of interestis an antibody, especially a monoclonal antibody. The antibody may befrom any mammal, such as a mouse, rat, rabbit, guinea pig, sheep, cow,or a transgenic animal with a human immunoglobulin locus, or it may be ahumanized, resurfaced, T-cell epitope depleted variant thereof.

It has to be understood that a person skilled in the art can practicethe method as reported herein to produce any polypeptide. Thus, anantibody as used herein has to be treated as an example of apolypeptide.

In the method as reported herein different antibody preparation can beused, such as non-purified (crude) or partially purified antibodies fromnatural, synthetic, or recombinant sources. Crude antibody preparationscan be obtained from various sources including, but not limited to,plasma, serum, ascites, or milk of an experimental animal, plantextracts, bacterial cell lysates, yeast lysates, or conditioned animalcell culture media. Partially purified preparations can be obtained fromcrude preparations that have been subjected to at least onechromatography, precipitation, other fractionation step, or anycombination thereof. The precipitation step can include any method, suchas salt or poly (ethylene glycol) precipitation. Other fractionationsteps such as crystallization or membrane filtration can also be used.In one embodiment the antibody is a not-PEGylated antibody.

With the method as reported herein a polypeptide can be obtained that issubstantially free of aggregates. In one embodiment is the content ofthe polypeptide in aggregated form as determined by size exclusionchromatography less than about 5% based on the total peak area. Inanother embodiment the content of the polypeptide in aggregated form isless than about 1%. In a further embodiment the amount of thepolypeptide in aggregated form is less than about 0.5%. In still anotherembodiment the amount of the polypeptide in aggregated form is less thanabout 0.1%.

Recently a new bispecific bivalent antibody format has been published(Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192), the so called“CrossMab”. This bispecific antibody comprises four different antibodychains, i.e. two different light chains and two different heavy chains.During the expression different product-related impurities can beformed. One of these product related impurities is the so called “¾antibody”, an antibody that is missing one light chain, displaying amolecular weight of about 125 kDa compared to a weight of about 150 kDafor a complete four chain antibody. These kinds of antibody-fragmentscan generally be formed during the expression of antibodies.

Thus, one aspect as reported herein is a method for producing a fulllength four chain antibody comprising the following steps:

-   -   a) cultivating a mammalian cell comprising a nucleic acid        encoding the antibody and recovering the antibody from the cell        or the cultivation medium,    -   b) purifying the antibody with a cation exchange chromatography        method comprising the following steps:        -   applying a solution comprising a non-ionic polymer and an            additive to a chromatography material to which an antibody            had been adsorbed, whereby the four chain full length            antibody in monomeric form remains adsorbed to an ion            exchange chromatography material, and        -   recovering the four chain full length antibody in monomeric            form from the ion exchange chromatography material by            applying a solution comprising a non-ionic polymer, an            additive, and an elution compound, and thereby producing the            antibody.

In one embodiment of all aspects as outlined before the ion exchangechromatography material is a cation exchange chromatography material.

In one embodiment of all aspects as outlined before the non-ionicpolymer is selected from the group comprising poly (ethylene glycol)(PEG), poly (propylene glycol) (PPG), PEG-PPG copolymers, and triblockcopolymers composed of poly (oxypropylene) (poly (propylene oxide))flanked by poly (oxyethylene) (poly (ethylene oxide)). In one embodimentthe non-ionic polymer is poly (ethylene glycol).

In one embodiment of all aspects as outlined before the additive isselected from the group comprising zwitterions, amino acids, urea, ureaderivatives, ampholytes, CHAPSO, natural products, sugars, and polyols.In a further embodiment the additive is sorbitol.

In one embodiment of all aspects as outlined before the non-ionicpolymer has a concentration of from about 5% to 15% by weight and/or theadditive has a concentration of from 3% to 25% by weight.

In one embodiment of all aspects as outlined before the polypeptide isan antibody of class IgG, or IgD, or IgE, or IgA. In another embodimentthe polypeptide is an antibody of class IgG, subclass IgG1, or subclassIgG2, or subclass IgG3, or subclass IgG4.

In one embodiment of all aspects as outlined before the method comprisesthe following steps:

-   -   applying a first solution that optionally comprises a non-ionic        polymer and an additive to an ion exchange chromatographic        material and thereby equilibrating the ion exchange        chromatography material,    -   applying a buffered solution comprising the polypeptide to the        equilibrated chromatography material and thereby adsorbing the        polypeptide to the chromatography material, whereby the solution        is essentially free of a non-ionic polymer and an additive,    -   applying a buffered solution comprising a non-ionic polymer and        an additive to the chromatography material, whereby the        polypeptide in monomeric form remains adsorbed to the ion        exchange chromatography material, and    -   recovering the polypeptide in monomeric form from the ion        exchange chromatography material by applying a solution        comprising a non-ionic polymer, an additive, and an elution        compound. In one embodiment the recovering is by gradient        elution.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

FIGURES

FIG. 1 Elution chromatogram of the cation exchange chromatography of theanti-IL17 antibody on the cation exchange chromatography material.

FIG. 2 Distribution of antibody in monomeric form and antibody inaggregated form over the collected fractions.

FIG. 3 Solubility of an anti-IL17 antibody and an anti-CSF-1R antibodyin a solution comprising poly (ethylene glycol) 4000 at a concentrationof 10% by weight as an example of a non-ionic polymer and differentpolyols at varying concentration as additive.

FIG. 4 Solubility of an anti-IL17 antibody and an anti-CSF-1R antibodyin a solution comprising poly (ethylene glycol) 4000 at a concentrationof 10% by weight as an example of a non-ionic polymer and differentamino acids at varying concentration as additive.

FIG. 5 Solubility of an anti-IL17 antibody and an anti-CSF-1R antibodyin a solution comprising poly (ethylene glycol) at a concentration of10% by weight as an example of a non-ionic polymer and different sugarsat varying concentration as additive.

FIG. 6 Distribution of antibody in monomeric form, ¾ antibody fragmentand antibody in aggregated form over the collected fractions forexamples 25 and 26.

MATERIAL

Each antibody (anti-IL17 antibody: for sequences see WO 2010/034443,anti-CSF-1R antibody: for sequences see PCT/EP2010/069090;anti-Ang2/VEGF antibody: for sequences see WO 2010/040508) was purifiedin a first step with a protein A affinity chromatography. Elution fromthe protein A column was carried out under acidic conditions. Afterwardsthe sample was adjusted to about pH 3.5 with 1 M citric acid andincubated for ˜1 hour. Subsequently the pH was adjusted to 5.0 or 5.5 byadding 1 M TRIS/HCl, pH 8-9 or 1 M TRIS solution (TRIS:tris-hydroxymethyl-amino-methane). After incubation at 4° C. for 12-18hours the sample was depth filtrated. The sample is a solution with aprotein concentration between 5 mg/ml and 20 mg/ml. This material isreferred to in the following as conditioned protein A eluate, which isprepared for loading onto a cation exchange chromatography material.

The anti-Ang2/VEGF antibody was in addition purified by anion exchangechromatography: The pH value of the conditioned protein A eluate wasadjusted to 7.5 by the addition of 1M TRIS buffer solution. Subsequentlythe sample was processed by anion exchange chromatography at pH 7.5 inflow-through mode.

In the first example the method as reported herein has been carried out.Thus, in Example 1 all conditions of the method are given. In thethereafter following examples only the differences are listed. All notmentioned parameters are identical.

Example 1 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Exchange material: Poros 50HS-   Column: 8 mm internal diameter, 100 mm length, 5.03 ml volume-   Flow rate: 90 cm/h (=0.75 ml/min)-   Equilibration solution: 10 mM sodium citrate buffer, adjusted to pH    5.0, 4 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution 1: 10 mM sodium citrate buffer, adjusted to pH 5.0, 1    column volume-   Wash solution 2: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol, adjusted to    pH 5.0-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol, comprising    750 mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The conditioned protein A eluate was applied to a chromatography columncontaining the ion exchange chromatography material. After the loadingstep at a flow rate of 90 cm/h the column was washed with wash solution1 (1 column volume) and wash solution 2 (3 column volumes). The antibodyin monomeric form was recovered with a linear gradient elution method,whereby the pH value was kept constant and the conductivity was varied(increased) by the addition of sodium chloride.

In FIG. 1 the elution chromatogram of the cation exchange chromatographyof the anti-IL17 antibody on the cation exchange chromatography materialis shown. The antibody in monomeric form and the antibody in aggregatedform are separated.

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration anti- antibody antibody (UV280) fraction bodymonomer aggregate [mg/ml] volume amount (SEC) (SEC) sample E = 1.45 [ml][mg] [%] [%] conditioned 8.93 10.7 95.67 93.35 6.65 protein A eluateflow-through 0.00 ~90 0.00 0 0 fraction 1 0.45 3.0 1.35 100 0 fraction 22.92 3.0 8.76 100 0 fraction 3 6.30 3.0 18.90 100 0 fraction 4 8.45 3.025.35 100 0 fraction 5 6.50 3.0 19.50 99.61 0.39 fraction 6 2.21 3.06.63 89.70 10.30 fraction 7 1.28 3.0 3.84 53.47 46.53 fraction 8 1.413.0 4.23 36.58 63.42 fraction 9 0.97 3.0 2.91 30.93 69.07 fraction 100.45 3.0 1.35 18.96 81.04 fraction 11 0.20 3.0 0.60 20.38 79.62 amountof CHO amount of CHO HCP based on amount of HCDNA based amount of amountof CHO on amount of CHO antibody HCP antibody HCDNA sample [ng/mg] [ng][pg/mg] [pg] conditioned 471 45,061 0.80 76.54 protein A eluateflow-through <5 ng/ml <15 <0.4 pg/ml <1.2 fraction 1 <18 <24 <9 <12.15fraction 2 <2.74 <24 <1 <8.76 fraction 3 <1.27 <24 <0.63 <11.91 fraction4 0.991 25 <0.96 <24.34 fraction 5 1.394 27 1 19.50 fraction 6 7.923 536 39.78 fraction 7 19 73 10 38.40 fraction 8 14 59 10 42.30 fraction 930 87 15 43.65 fraction 10 91 123 26 35.10 fraction 11 316 190 53 31.80

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.78.

Example 2 Chromatography Conditions

-   Polypeptide: anti-CSF-1R antibody

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content. The result of theanalysis is presented in the following Table.

TABLE concentration anti- antibody antibody (UV280) fraction bodymonomer aggregate [mg/ml] volume amount (SEC) (SEC) sample E = 1.47 [ml][mg] [%] [%] conditioned 12.9 7.71 99.48 99.19 0.81 protein A eluateflow-through 0.00 ~60 0.00 — — fraction 1 0.99 3.00 2.97 100 0 fraction2 5.84 3.00 17.52 100 0 fraction 3 8.30 3.00 24.90 100 0 fraction 4 7.873.00 23.61 100 0 fraction 5 5.67 3.00 17.01 100 0 fraction 6 1.94 3.005.82 100 0 fraction 7 0.58 3.00 1.74 94.21 5.79 fraction 8 0.41 3.001.23 87.79 12.21 fraction 9 0.23 1.50 0.35 84.58 15.42 amount of CHOamount of CHO HCP based on amount of HCDNA based amount of amount of CHOon amount of CHO antibody HCP antibody HCDNA sample [ng/mg] [ng] [pg/mg][pg] conditioned 1,260 125,345 2 198.96 protein A eluate flow-through <5ng/ml <15 <0.4 pg/ml <1.2 fraction 1 <8.081 <24 <4 11.88 fraction 21.579 28 <0.68 70.08 fraction 3 <0.602 <15 <0.48 16.93 fraction 4 <1.017<24 1 11.33 fraction 5 2.797 48 1 17.01 fraction 6 14 81 5 5.82 fraction7 50 87 19 8.70 fraction 8 99 122 28 23.37 fraction 9 n.a. n.a. n.a.n.a.

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.88.

Example 3 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Equilibration solution: 35 mM sodium acetate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol adjusted to pH    5.0, 4 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 35 mM sodium acetate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol adjusted to pH 5.0, 1    column volume-   Elution solution: 35 mM sodium acetate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol, comprising    750 mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration anti- antibody antibody (UV280) fraction bodymonomer aggregate [mg/ml] volume amount (SEC) (SEC) sample E = 1.45 [ml][mg] [%] [%] conditioned 9.36 11.0 102.98 92.76 7.24 protein A eluateflow-through 0.00 ~50 0.00 0 0 fraction 1 0.43 2.0 0.86 100 0 fraction 22.77 2.0 5.54 100 0 fraction 3 6.10 2.0 12.20 100 0 fraction 4 8.02 2.016.04 100 0 fraction 5 8.56 2.0 17.12 100 0 fraction 6 7.86 2.0 15.72100 0 fraction 7 5.59 2.0 11.18 99.77 0.23 fraction 8 2.74 2.0 5.4897.75 2.25 fraction 9 1.34 2.0 2.68 85.98 14.02 fraction 10 1.13 2.02.26 55.26 44.74 fraction 11 1.31 2.0 2.62 35.5 64.5 fraction 12 1.412.0 2.82 24.86 75.14 fraction 13 1.18 2.0 2.36 22.22 77.78 fraction 140.79 2.0 1.58 15.47 84.53 fraction 15 0.48 2.0 0.96 13.63 86.37 fraction16 0.29 2.0 0.58 13.73 86.27 fraction 17 0.18 2.0 0.36 16.03 83.97

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.86.

Example 4 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Equilibration solution: 25 mM MES buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol adjusted to pH 5.5, 4    column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 35 mM MES buffer, 10% (w/w) poly (ethylene glycol)    (MW 3,500 Da), 15% (w/w) D-sorbitol adjusted to pH 5.5, 4 column    volumes-   Elution solution: 35 mM MES buffer, 10% (w/w) poly (ethylene glycol)    (MW 3,500 Da), 15% (w/w) D-sorbitol, comprising 750 mM sodium    chloride, adjusted to pH 5.5-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration anti- antibody antibody (UV280) fraction bodymonomer aggregate [mg/ml] volume amount (SEC) (SEC) sample E = 1.45 [ml][mg] [%] [%] conditioned 9.36 10.99 102.87 92.85 7.15 protein A eluateflow-through 0.00 ~50 0.00 0 0 fraction 1 0.28 2.0 0.56 100 0 fraction 21.45 2.0 2.90 100 0 fraction 3 4.43 2.0 8.86 100 0 fraction 4 7.17 2.014.34 100 0 fraction 5 8.90 2.0 17.80 100 0 fraction 6 8.85 2.0 17.70100 0 fraction 7 6.64 2.0 13.28 99.65 0.35 fraction 8 2.97 2.0 5.9493.13 6.87 fraction 9 1.61 2.0 3.22 67.63 32.37 fraction 10 1.50 2.03.00 34.08 65.92 fraction 11 1.51 2.0 3.02 25.22 74.78 fraction 12 1.192.0 2.38 22.68 77.32 fraction 13 0.72 2.0 1.44 20.45 79.55 fraction 140.43 2.0 0.86 17.36 82.64 fraction 15 0.25 2.0 0.50 16.24 83.76 fraction16 0.15 2.0 0.30 18.61 81.39

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.77.

Example 5 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Exchange material: SP Sepharose FF-   Equilibration solution: 30 mM sodium citrate buffer, adjusted to pH    5.0, 4 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution 1: 30 mM sodium citrate buffer, adjusted to pH 5.0, 1    column volume-   Wash solution 2: 30 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol, adjusted to    pH 5.0-   Elution solution: 30 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol, comprising    750 mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration anti- antibody antibody (UV280) fraction bodymonomer aggregate [mg/ml] volume amount (SEC) (SEC) sample E = 1.45 [ml][mg] [%] [%] conditioned 9.36 10.7 100.07 92.90 7.10 protein A eluateflow-through 0.00 ~50 0.00 — — fraction 1 0.18 2.00 0.36 100 0 fraction2 0.74 2.00 1.48 100 0 fraction 3 1.97 2.00 3.94 100 0 fraction 4 3.622.00 7.24 100 0 fraction 5 5.15 2.00 10.30 100 0 fraction 6 6.17 2.0012.34 99.65 0.35 fraction 7 6.25 2.00 12.50 99.23 0.77 fraction 8 5.642.00 11.28 98.11 1.89 fraction 9 4.53 2.00 9.06 96.18 3.82 fraction 103.34 2.00 6.68 92.74 7.26 fraction 11 2.37 2.00 4.74 85.49 14.51fraction 12 1.63 2.00 3.26 77.02 22.98 fraction 13 1.15 2.00 2.30 63.2736.73 fraction 14 0.83 2.00 1.66 48.97 51.03 fraction 15 0.60 2.00 1.2036.01 63.99 fraction 16 0.44 2.00 0.88 25.04 74.96 fraction 17 0.32 2.000.64 19.77 80.23 fraction 18 0.23 2.00 0.46 17.00 83.00 fraction 19 0.162.00 0.32 16.62 83.38

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.51.

Example 6 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Exchange material: Toyopearl CM-650 M-   Equilibration solution: 30 mM sodium citrate buffer, adjusted to pH    5.0, 4 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution 1: 30 mM sodium citrate buffer, adjusted to pH 5.0, 1    column volume-   Wash solution 2: 30 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol, adjusted to    pH 5.0-   Elution solution: 30 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol, comprising    750 mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration anti- antibody antibody (UV280) fraction bodymonomer aggregate [mg/ml] volume amount (SEC) (SEC) sample E = 1.45 [ml][mg] [%] [%] conditioned 9.38 10.7 100.32 93.05 6.95 protein A eluateflow-through 0.00 ~50 0.00 0 0 fraction 1 0.58 2.00 1.16 100 0 fraction2 1.63 2.00 3.26 100 0 fraction 3 2.89 2.00 5.78 100 0 fraction 4 4.132.00 8.26 100 0 fraction 5 5.01 2.00 10.02 100 0 fraction 6 5.49 2.0010.98 100 0 fraction 7 5.58 2.00 11.16 100 0 fraction 8 5.24 2.00 10.48100 0 fraction 9 4.52 2.00 9.04 100 0 fraction 10 3.48 2.00 6.96 99.290.71 fraction 11 2.41 2.00 4.82 97.27 2.73 fraction 12 1.58 2.00 3.1691.13 8.87 fraction 13 1.15 2.00 2.30 75.73 24.27 fraction 14 0.99 2.001.98 44.16 55.84 fraction 15 0.94 2.00 1.88 17.21 82.79 fraction 16 0.882.00 1.76 16.96 83.04 fraction 17 0.75 2.00 1.50 10.07 89.93 fraction 180.58 2.00 1.16 8.64 91.36 fraction 19 0.43 2.00 0.86 0.85 99.15 fraction20 0.31 2.00 0.62 0 100 fraction 21 0.21 2.00 0.42 0 100 fraction 220.15 2.00 0.30 0 100

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.76.

Example 7 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Exchange material: CM Sepharose FF-   Equilibration solution: 30 mM sodium citrate buffer, adjusted to pH    5.0, 4 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution 1: 30 mM sodium citrate buffer, adjusted to pH 5.0, 1    column volume-   Wash solution 2: 30 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol, adjusted to    pH 5.0-   Elution solution: 30 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 15% (w/w) D-sorbitol, comprising    750 mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration anti- antibody antibody (UV280) fraction bodymonomer aggregate [mg/ml] volume amount (SEC) (SEC) sample E = 1.45 [ml][mg] [%] [%] conditioned 9.55 10.7 102.02 93.04 6.96 protein A eluateflow-through 0.00 ~50 0.00 — — fraction 1 0.14 2.00 0.28 98.77 1.23fraction 2 0.47 2.00 0.94 100 0 fraction 3 1.16 2.00 2.32 100 0 fraction4 2.19 2.00 4.38 100 0 fraction 5 3.32 2.00 6.64 100 0 fraction 6 4.382.00 8.76 100 0 fraction 7 4.98 2.00 9.96 99.79 0.21 fraction 8 5.372.00 10.74 99.48 0.52 fraction 9 5.18 2.00 10.36 98.65 1.35 fraction 104.61 2.00 9.22 98.21 1.79 fraction 11 3.89 2.00 7.78 96.20 3.80 fraction12 3.01 2.00 6.02 93.77 6.23 fraction 13 2.35 2.00 4.70 89.37 10.63fraction 14 1.76 2.00 3.52 83.97 16.03 fraction 15 1.30 2.00 2.60 75.3924.61 fraction 16 0.98 2.00 1.96 63.61 36.39 fraction 17 0.74 2.00 1.4851.68 48.32 fraction 18 0.57 2.00 1.14 37.62 62.38 fraction 19 0.44 2.000.88 30.42 69.58 fraction 20 0.33 2.00 0.66 22.95 77.05 fraction 21 0.232.00 0.46 21.01 78.99 fraction 22 0.19 2.00 0.38 16.76 83.24 fraction 230.15 2.00 0.30 15.29 84.71

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.51.

Example 8 Chromatography Conditions

-   Polypeptide: anti-IL 17 antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 20% (w/w) xylitol adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 20% (w/w) xylitol adjusted to pH 5.0, 4    column volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 20% (w/w) xylitol, comprising 750    mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.45 [ml] [mg] [%] [%] [%] conditioned 9.36 10.77 100.2494.23 5.77 0 protein A eluate flow-through 0.00 ~80 0.00 — — 0 fraction1 0.62 1.5 0.93 82.66 0 17.34 fraction 2 2.85 1.5 4.28 100 0 0 fraction3 5.59 1.5 8.39 100 0 0 fraction 4 7.15 1.5 10.73 100 0 0 fraction 57.89 1.5 11.84 100 0 0 fraction 6 7.81 1.5 11.72 100 0 0 fraction 7 7.311.5 10.97 100 0 0 fraction 8 6.21 1.5 9.32 100 0 0 fraction 9 4.48 1.56.72 99.78 0.22 0 fraction 10 2.74 1.5 4.11 98.36 1.64 0 fraction 111.65 1.5 2.48 94.44 5.56 0 fraction 12 1.24 1.5 1.86 77.49 22.51 0fraction 13 1.23 1.5 1.85 57.48 42.52 0 fraction 14 1.39 1.5 2.09 43.2756.73 0 fraction 15 1.43 1.5 2.15 36.42 63.58 0 fraction 16 1.30 1.51.95 34.12 65.88 0 fraction 17 1.01 1.5 1.52 29.98 70.02 0 fraction 180.72 1.5 1.08 23.98 76.02 0 fraction 19 0.48 1.5 0.72 26.01 73.99 0fraction 20 0.32 1.5 0.48 20.98 79.02 0 fraction 21 0.22 1.5 0.33 20.2379.77 0 fraction 22 0.15 1.5 0.23 21.64 78.36 0

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.76.

Example 9 Chromatography Conditions

-   Polypeptide: anti-CSF-1R antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 20% (w/w) xylitol adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 20% (w/w) xylitol adjusted to pH 5.0, 4    column volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 20% (w/w) xylitol, comprising 750    mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.47 [ml] [mg] [%] [%] [%] conditioned 13.3 7.41 98.58 99.190.81 0 protein A eluate flow-through 0.00 ~80 0.00 0 0 0 fraction 1 1.741.5 2.61 100 0 0 fraction 2 4.90 1.5 7.35 100 0 0 fraction 3 6.98 1.510.47 100 0 0 fraction 4 7.83 1.5 11.75 100 0 0 fraction 5 7.96 1.511.94 100 0 0 fraction 6 7.69 1.5 11.54 100 0 0 fraction 7 7.20 1.510.80 100 0 0 fraction 8 6.37 1.5 9.56 100 0 0 fraction 9 4.73 1.5 7.10100 0 0 fraction 10 3.14 1.5 4.71 100 0 0 fraction 11 1.78 1.5 2.6799.57 0.43 0 fraction 12 0.89 1.5 1.34 98.06 1.94 0 fraction 13 0.64 1.50.96 91.40 8.60 0 fraction 14 0.57 1.5 0.86 70.19 21.01 8.80 fraction 150.55 1.5 0.83 63.04 28.85 8.11 fraction 16 0.43 1.5 0.65 58.67 31.489.85 fraction 17 0.28 1.5 0.42 57.19 34.17 8.64

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.93.

Example 10 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 30% (w/w) glycerol adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 30% (w/w) glycerol adjusted to pH 5.0, 4    column volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 30% (w/w) glycerol, comprising 750    mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.45 [ml] [mg] [%] [%] [%] conditioned 9.36 10.69 100.0493.93 6.07 0 protein A eluate flow-through 0.00 ~80 0.00 0 0 0 fraction1 0.92 1.5 1.38 66.77 0 33.23 fraction 2 2.90 1.5 4.35 100 0 0 fraction3 5.06 1.5 7.59 100 0 0 fraction 4 6.36 1.5 9.54 100 0 0 fraction 5 6.711.5 10.07 100 0 0 fraction 6 6.74 1.5 10.11 100 0 0 fraction 7 6.45 1.59.68 100 0 0 fraction 8 5.71 1.5 8.57 100 0 0 fraction 9 4.70 1.5 7.0599.84 0.16 0 fraction 10 3.50 1.5 5.25 99.54 0.46 0 fraction 11 2.40 1.53.60 98.34 1.66 0 fraction 12 1.61 1.5 2.42 94.80 5.20 0 fraction 131.23 1.5 1.85 86.37 13.63 0 fraction 14 1.16 1.5 1.74 66.30 33.70 0fraction 15 1.28 1.5 1.92 48.88 51.12 0 fraction 16 1.38 1.5 2.07 30.9669.04 0 fraction 17 1.34 1.5 2.01 29.30 70.70 0 fraction 18 0.88 1.51.32 30.99 69.01 0 fraction 19 0.89 1.5 1.34 27.69 72.33 0 fraction 200.64 1.5 0.96 31.36 68.64 0 fraction 21 0.56 1.5 0.84 30.76 69.24 0fraction 22 0.35 1.5 0.53 29.23 70.77 0 fraction 23 0.25 1.5 0.38 27.8372.17 0 fraction 24 0.18 1.5 0.27 27.50 72.50 0

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.77.

Example 11 Chromatography Conditions

-   Polypeptide: anti-CSF-1R antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 30% (w/w) glycerol adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 30% (w/w) glycerol adjusted to pH 5.0, 4    column volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 30% (w/w) glycerol, comprising 750    mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.47 [ml] [mg] [%] [%] [%] conditioned 13.3 7.55 100.64 99.130.87 0 protein A eluate flow-through 0.00 ~80 0.00 0 0 0 fraction 1 1.271.5 1.91 81.59 0 18.41 fraction 2 3.61 1.5 5.42 100 0 0 fraction 3 5.451.5 8.18 100 0 0 fraction 4 6.36 1.5 9.54 100 0 0 fraction 5 6.48 1.59.72 100 0 0 fraction 6 6.50 1.5 9.75 100 0 0 fraction 7 6.28 1.5 9.42100 0 0 fraction 8 5.79 1.5 8.69 100 0 0 fraction 9 5.17 1.5 7.76 100 00 fraction 10 4.25 1.5 6.38 100 0 0 fraction 11 3.26 1.5 4.89 100 0 0fraction 12 2.27 1.5 3.41 99.85 0.15 0 fraction 13 1.43 1.5 2.15 93.210.35 6.45 fraction 14 0.86 1.5 1.29 69.58 1.24 29.18 fraction 15 0.601.5 0.90 58.01 5.92 36.07 fraction 16 0.51 1.5 0.77 51.57 15.49 32.94fraction 17 0.50 1.5 0.75 46.54 26.33 27.14 fraction 18 0.45 1.5 0.6843.00 31.02 25.97 fraction 19 0.43 1.5 0.65 36.82 38.46 24.72 fraction20 0.27 1.5 0.41 37.11 38.01 24.87

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.90.

Example 12 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 25% (w/w) saccharose adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 25% (w/w) saccharose adjusted to pH 5.0, 4    column volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 25% (w/w) saccharose, comprising    750 mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.45 [ml] [mg] [%] [%] [%] conditioned 9.36 10.71 96.91 94.975.03 0 protein A eluate flow-through 0.00 ~90 0.00 0 0 0 fraction 1 1.121.5 1.68 93.40 0 6.60 fraction 2 3.57 1.5 5.36 100 0 0 fraction 3 6.051.5 9.08 100 0 0 fraction 4 7.44 1.5 11.16 100 0 0 fraction 5 7.8 1.511.70 100 0 0 fraction 6 7.64 1.5 11.46 100 0 0 fraction 7 6.96 1.510.44 100 0 0 fraction 8 5.89 1.5 8.84 100 0 0 fraction 9 4.35 1.5 6.5399.82 0.18 0 fraction 10 2.85 1.5 4.28 98.95 1.05 0 fraction 11 1.81 1.52.72 95.18 4.82 0 fraction 12 1.33 1.5 2.00 85.83 14.17 0 fraction 131.23 1.5 1.85 70.44 29.56 0 fraction 14 1.26 1.5 1.89 59.07 40.93 0fraction 15 1.2 1.5 1.80 50.95 49.05 0 fraction 16 1.01 1.5 1.52 43.6756.33 0 fraction 17 0.77 1.5 1.16 42.33 57.67 0 fraction 18 0.53 1.50.80 36.74 63.26 0 fraction 19 0.36 1.5 0.54 37.68 62.32 0 fraction 200.25 1.5 0.38 36.00 64.00 0 fraction 21 0.18 1.5 0.27 34.41 65.59 0

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.73.

Example 13 Chromatography Conditions

-   Polypeptide: anti-CSF-1R antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 25% (w/w) saccharose adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 25% (w/w) saccharose adjusted to pH 5.0, 4    column volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 25% (w/w) saccharose, comprising    750 mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.47 [ml] [mg] [%] [%] [%] conditioned 13.8 7.55 100.44 99.230.77 0 protein A eluate flow-through 0.00 ~90 0.00 0 0 0 fraction 1 1.781.5 2.67 100 0 0 fraction 2 4.54 1.5 6.81 100 0 0 fraction 3 6.45 1.59.68 100 0 0 fraction 4 7.46 1.5 11.19 100 0 0 fraction 5 7.50 1.5 11.25100 0 0 fraction 6 7.35 1.5 11.03 100 0 0 fraction 7 6.79 1.5 10.19 1000 0 fraction 8 6.01 1.5 9.02 100 0 0 fraction 9 4.87 1.5 7.31 100 0 0fraction 10 3.63 1.5 5.45 100 0 0 fraction 11 2.38 1.5 3.57 100 0 0fraction 12 1.41 1.5 2.12 99.73 0.27 0 fraction 13 0.84 1.5 1.26 98.711.29 0 fraction 14 0.60 1.5 0.90 83.46 5.09 11.45 fraction 15 0.52 1.50.78 74.12 9.89 15.99 fraction 16 0.44 1.5 0.66 71.45 11.73 16.82fraction 17 0.33 1.5 0.50 70.55 13.67 15.77 fraction 18 0.22 1.5 0.3361.41 14.38 24.21

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.84.

Example 14 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 25% (w/w) D-fructose adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 25% (w/w) D-fructose adjusted to pH 5.0, 4    column volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 25% (w/w) D-fructose, comprising    750 mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.45 [ml] [mg] [%] [%] [%] conditioned 9.36 10.7 100.18 95.114.89 0 protein A eluate flow-through 0.00 ~80 0.00 0 0 0 fraction 1 0.931.5 1.40 79.49 0 20.51 fraction 2 3.94 1.5 5.91 100 0 0 fraction 3 6.681.5 10.02 100 0 0 fraction 4 7.86 1.5 11.79 100 0 0 fraction 5 7.98 1.511.97 100 0 0 fraction 6 7.52 1.5 11.28 100 0 0 fraction 7 6.83 1.510.25 100 0 0 fraction 8 5.75 1.5 8.63 100 0 0 fraction 9 4.45 1.5 6.6899.78 0.22 0 fraction 10 3.08 1.5 4.62 99.45 0.55 0 fraction 11 1.97 1.52.96 97.64 2.36 0 fraction 12 1.39 1.5 2.09 92.02 7.98 0 fraction 131.06 1.5 1.59 78.43 21.57 0 fraction 14 1.13 1.5 1.70 59.36 40.64 0fraction 15 1.24 1.5 1.86 44.05 55.95 0 fraction 16 1.18 1.5 1.77 37.2062.80 0 fraction 17 1.08 1.5 1.62 32.47 67.53 0 fraction 18 0.76 1.51.14 29.88 70.12 0 fraction 19 0.67 1.5 1.01 35.55 64.45 0 fraction 200.41 1.5 0.62 28.90 71.10 0 fraction 21 0.29 1.5 0.44 25.31 74.69 0fraction 22 0.21 1.5 0.32 23.60 76.40 0 fraction 23 0.20 1.5 0.30 46.1653.84 0

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.79.

Example 15 Chromatography Conditions

-   Polypeptide: anti-CSF-1R antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 25% (w/w) D-fructose adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 25% (w/w) D-fructose adjusted to pH 5.0, 4    column volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 25% (w/w) D-fructose, comprising    750 mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.47 [ml] [mg] [%] [%] [%] conditioned 13.3 7.55 100.43 99.220.78 0 protein A eluate flow-through 0.00 ~80 0.00 0 0 0 fraction 1 0.541.5 0.81 77.09 0.22 22.69 fraction 2 2.58 1.5 3.87 100 0 0 fraction 35.32 1.5 7.98 100 0 0 fraction 4 7.29 1.5 10.94 100 0 0 fraction 5 7.781.5 11.67 100 0 0 fraction 6 7.96 1.5 11.94 100 0 0 fraction 7 7.72 1.511.58 100 0 0 fraction 8 6.87 1.5 10.31 100 0 0 fraction 9 6.10 1.5 9.15100 0 0 fraction 10 4.87 1.5 7.31 100 0 0 fraction 11 3.61 1.5 5.42 1000 0 fraction 12 2.17 1.5 3.26 99.89 0.11 0 fraction 13 1.27 1.5 1.9199.24 0.76 0 fraction 14 0.81 1.5 1.22 89.35 4.06 6.58 fraction 15 0.631.5 0.95 80.84 11.62 7.54 fraction 16 0.59 1.5 0.89 72.37 20.76 6.86fraction 17 0.50 1.5 0.75 66.31 25.98 7.71 fraction 18 0.40 1.5 0.6064.08 29.11 6.81 fraction 19 0.26 1.5 0.39 61.91 31.23 6.86 fraction 200.17 1.5 0.26 58.82 32.78 8.41

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.90.

Example 16 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 8% (w/w) glycine adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 8% (w/w) glycine adjusted to pH 5.0, 4 column    volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 8% (w/w) glycine, comprising 750 mM    sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.45 [ml] [mg] [%] [%] [%] conditioned 8.82 10.76 94.87 94.885.12 0 protein A eluate flow-through 0.00 ~90 0.00 0 0 0 fraction 1 0.311.5 0.47 53.77 0 46.73 fraction 2 1.57 1.5 2.36 100 0 0 fraction 3 4.681.5 7.02 100 0 0 fraction 4 7.51 1.5 11.27 100 0 0 fraction 5 9.34 1.514.01 100 0 0 fraction 6 9.92 1.5 14.88 100 0 0 fraction 7 9.60 1.514.40 100 0 0 fraction 8 7.02 1.5 10.53 99.64 0.36 0 fraction 9 3.08 1.54.62 97.59 2.41 0 fraction 10 1.50 1.5 2.25 88.29 11.71 0 fraction 111.33 1.5 2.00 70.61 29.39 0 fraction 12 1.45 1.5 2.18 58.23 41.77 0fraction 13 1.32 1.5 1.98 50.02 49.98 0 fraction 14 0.86 1.5 1.29 43.8556.15 0 fraction 15 0.47 1.5 0.71 42.73 57.27 0 fraction 16 0.25 1.50.38 38.54 61.46 0

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.85.

Example 17 Chromatography Conditions

-   Polypeptide: anti-CSF-1R antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 8% (w/w) glycine adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 8% (w/w) glycine adjusted to pH 5.0, 4 column    volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 8% (w/w) glycine, comprising 750 mM    sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.47 [ml] [mg] [%] [%] [%] conditioned 13.3 6.87 99.16 99.280.72 0 protein A eluate flow-through 0.00 ~90 0.00 0 0 0 fraction 1 0.281.5 0.42 66.36 0.41 33.23 fraction 2 1.82 1.5 2.73 100 0 0 fraction 35.02 1.5 7.53 100 0 0 fraction 4 7.74 1.5 11.61 100 0 0 fraction 5 9.051.5 13.58 100 0 0 fraction 6 9.19 1.5 13.79 100 0 0 fraction 7 8.67 1.513.01 100 0 0 fraction 8 7.04 1.5 10.56 100 0 0 fraction 9 3.82 1.5 5.7399.90 0.10 0 fraction 10 1.46 1.5 2.19 99.00 1.00 0 fraction 11 0.70 1.51.05 95.61 4.39 0 fraction 12 0.56 1.5 0.84 84.11 6.96 8.92 fraction 130.42 1.5 0.63 79.56 8.46 11.99 fraction 14 0.25 1.5 0.38 72.00 12.7115.29

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.90.

Example 18 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 8% (w/w) L-proline adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 8% (w/w) L-proline adjusted to pH 5.0, 4    column volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 8% (w/w) L-proline, comprising 750    mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.45 [ml] [mg] [%] [%] [%] conditioned 9.36 10.6 93.51 94.975.03 0 protein A eluate flow-through 0.00 ~90 0.00 0 0 0 fraction 1 0.261.5 0.39 58.65 0 41.35 fraction 2 0.95 1.5 1.43 100 0 0 fraction 3 2.581.5 3.87 100 0 0 fraction 4 4.75 1.5 7.13 100 0 0 fraction 5 6.24 1.59.36 100 0 0 fraction 6 7.22 1.5 10.83 100 0 0 fraction 7 7.96 1.5 11.94100 0 0 fraction 8 7.97 1.5 11.96 100 0 0 fraction 9 7.35 1.5 11.03 1000 0 fraction 10 5.76 1.5 8.64 100 0 0 fraction 11 2.89 1.5 4.34 98.951.05 0 fraction 12 1.18 1.5 1.77 94.31 5.69 0 fraction 13 0.68 1.5 1.0280.42 19.58 0 fraction 14 0.63 1.5 0.95 68.26 31.74 0 fraction 15 0.721.5 1.08 50.87 49.13 0 fraction 16 0.84 1.5 1.26 60.95 39.05 0 fraction17 0.90 1.5 1.35 75.25 24.75 0 fraction 18 0.81 1.5 1.22 78.25 21.75 0fraction 19 0.59 1.5 0.89 79.58 20.42 0 fraction 20 0.38 1.5 0.57 73.7626.24 0 fraction 21 0.24 1.5 0.36 75.45 24.55 0 fraction 22 0.16 1.50.24 76.72 23.28 0

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.83.

Example 19 Chromatography Conditions

Polypeptide: anti-CSF-1R antibody

Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly(ethylene glycol) (MW 3,500 Da), 8% (w/w) L-proline adjusted to pH 5.0,2 column volumes

Loading: 20 g protein/1 chromatography material

Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethyleneglycol) (MW 3,500 Da), 8% (w/w) L-proline adjusted to pH 5.0, 4 columnvolume

Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethyleneglycol) (MW 3,500 Da), 8% (w/w) L-proline, comprising 750 mM sodiumchloride, adjusted to pH 5.0

Elution method: linear gradient from 0% (v/v) to 100% (v/v) elutionsolution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.47 [ml] [mg] [%] [%] [%] conditioned 13.3 7.31 97.20 99.330.67 0 protein A eluate flow-through 0.00 ~90 0.00 0 0 0 fraction 1 0.511.5 0.77 67.91 0.38 31.71 fraction 2 2.31 1.5 3.47 100 0 0 fraction 34.90 1.5 7.35 100 0 0 fraction 4 6.60 1.5 9.90 100 0 0 fraction 5 7.411.5 11.12 100 0 0 fraction 6 7.70 1.5 11.55 100 0 0 fraction 7 7.87 1.511.81 100 0 0 fraction 8 7.21 1.5 10.82 100 0 0 fraction 9 6.14 1.5 9.21100 0 0 fraction 10 4.35 1.5 6.53 100 0 0 fraction 11 2.13 1.5 3.2099.89 0.11 0 fraction 12 0.83 1.5 1.25 99.16 0.84 0 fraction 13 0.38 1.50.57 79.11 5.30 15.59 fraction 14 0.27 1.5 0.41 63.62 16.62 19.76fraction 15 0.24 1.5 0.36 46.29 27.44 26.27 fraction 16 0.18 1.5 0.2768.51 31.49 20.66

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.97.

Example 20 Chromatography Conditions

-   Polypeptide: anti-IL17 antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 8% (w/w) Betaine adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 8% (w/w) Betaine adjusted to pH 5.0, 4 column    volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 8% (w/w) Betaine, comprising 750 mM    sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.45 [ml] [mg] [%] [%] [%] conditioned 9.24 10.81 99.86 94.165.84 0 protein A eluate flow-through 0.00 ~90 0.00 0 0 0 fraction 1 0.211.5 0.32 51.88 0 48.12 fraction 2 0.61 1.5 0.92 57.58 0 42.46 fraction 31.59 1.5 2.39 100 0 0 fraction 4 3.25 1.5 4.88 100 0 0 fraction 5 5.111.5 7.67 100 0 0 fraction 6 6.63 1.5 9.95 100 0 0 fraction 7 7.69 1.511.54 100 0 0 fraction 8 8.26 1.5 12.39 100 0 0 fraction 9 8.15 1.512.23 100 0 0 fraction 10 7.88 1.5 11.82 100 0 0 fraction 11 5.02 1.57.53 100 0 0 fraction 12 1.91 1.5 2.87 95.64 4.36 0 fraction 13 0.84 1.51.26 85.39 14.61 0 fraction 14 0.72 1.5 1.08 68.77 31.23 0 fraction 150.81 1.5 1.22 66.73 33.27 0 fraction 16 0.89 1.5 1.34 64.45 35.55 0fraction 17 0.88 1.5 1.32 49.18 50.82 0 fraction 18 0.77 1.5 1.16 46.3153.69 0 fraction 19 0.53 1.5 0.80 44.49 55.51 0 fraction 20 0.34 1.50.51 35.48 64.52 0 fraction 21 0.22 1.5 0.33 33.50 66.5 0 fraction 220.15 1.5 0.23 32.72 67.28 0

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.86.

Example 21 Chromatography Conditions

-   Polypeptide: anti-CSF-1R antibody-   Equilibration solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 8% (w/w) Betaine adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 3,500 Da), 8% (w/w) Betaine adjusted to pH 5.0, 4 column    volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 3,500 Da), 8% (w/w) Betaine, comprising 750 mM    sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration antibody antibody antibody (UV280) fraction antibodymonomer aggregate fragments [mg/ml] volume amount (SEC) (SEC) (SEC)sample E = 1.47 [ml] [mg] [%] [%] [%] conditioned 13.8 7.55 99.16 99.220.78 0 protein A eluate flow-through 0.00 ~90 0.00 0 0 0 fraction 1 0.331.5 0.50 71.12 0.40 28.48 fraction 2 1.61 1.5 2.42 100 0 0 fraction 34.31 1.5 6.47 100 0 0 fraction 4 6.58 1.5 9.87 100 0 0 fraction 5 7.751.5 11.63 100 0 0 fraction 6 8.30 1.5 12.45 100 0 0 fraction 7 8.40 1.512.60 100 0 0 fraction 8 8.13 1.5 12.20 100 0 0 fraction 9 7.34 1.511.01 100 0 0 fraction 10 5.40 1.5 8.10 100 0 0 fraction 11 2.61 1.53.92 99.82 0.18 0 fraction 12 0.92 1.5 1.38 98.56 1.44 0 fraction 130.42 1.5 0.63 92.33 7.67 0 fraction 14 0.34 1.5 0.51 88.25 11.75 0fraction 15 0.32 1.5 0.48 85.93 14.07 0 fraction 16 0.24 1.5 0.36 82.3817.62 0 fraction 17 0.17 1.5 0.26 77.09 22.91 0

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.86.

Example 22 Chromatography Conditions

-   Polypeptide: anti-IL 17 antibody-   Equilibration solution: 10 mM sodium citrate buffer adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 400 Da), 15% (w/w) sorbitol adjusted to pH 5.0, 4 column    volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 400 Da), 15% (w/w) sorbitol, comprising 750 mM    sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 22.5 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration anti- antibody antibody (UV280) fraction bodymonomer aggregate [mg/ml] volume amount (SEC) (SEC) sample E = 1.47 [ml][mg] [%] [%] conditioned 9.76 10.8 105.00 93.34 6.66 protein A eluateflow-through 0.00 ~60 0.00 — — fraction 1 3.19 2.0 6.38 100 0 fraction 211.2 2.0 22.40 100 0 fraction 3 12.2 2.0 24.40 100 0 fraction 4 8.89 2.017.78 99.64 0.36 fraction 5 5.22 2.0 10.44 94.65 5.35 fraction 6 3.452.0 6.90 72.24 27.76 fraction 7 2.33 2.0 4.66 44.63 55.37 fraction 81.46 2.0 2.92 33.75 66.25 fraction 9 0.86 2.0 1.72 24.38 75.62 fraction10 0.46 2.0 0.92 30.67 69.33 fraction 11 0.27 2.0 0.54 51.34 48.66fraction 12 0.20 2.0 0.40 64.8 35.2 fraction 13 0.17 2.0 0.34 71.5828.42 fraction 14 0.15 2.0 0.30 75.35 24.65

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.57.

Example 23 Chromatography Conditions

-   Polypeptide: anti-IL 17 antibody-   Equilibration solution: 10 mM sodium citrate buffer adjusted to pH    5.0, 2 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 10 mM sodium citrate buffer, 10% (w/w) poly (ethylene    glycol) (MW 10,000 Da), 15% (w/w) sorbitol adjusted to pH 5.0, 4    column volume-   Elution solution: 10 mM sodium citrate buffer, 10% (w/w) poly    (ethylene glycol) (MW 10,000 Da), 15% (w/w) sorbitol, comprising 750    mM sodium chloride, adjusted to pH 5.0-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 30 column volumes

The peak containing the antibody was collected in 3 ml fractions, whichwere analyzed for monomer and aggregate content as well as for CHO hostcell protein content and CHO host cell DNA content. The result of theanalysis is presented in the following Table.

TABLE concentration anti- antibody antibody (UV280) fraction bodymonomer aggregate [mg/ml] volume amount (SEC) (SEC) sample E = 1.47 [ml][mg] [%] [%] conditioned 9.06 10.5 95.13 93.30 6.70 protein A eluateflow-through 0.00 ~100 0.00 — — fraction 1 0.27 2.0 0.54 100 0 fraction2 0.56 2.0 1.12 100 0 fraction 3 1.04 2.0 2.08 100 0 fraction 4 1.59 2.03.18 100 0 fraction 5 2.09 2.0 4.18 100 0 fraction 6 2.46 2.0 4.92 100 0fraction 7 2.78 2.0 5.56 100 0 fraction 8 3.03 2.0 6.06 100 0 fraction 93.06 2.0 6.12 100 0 fraction 10 3.15 2.0 6.30 100 0 fraction 11 3.06 2.06.12 100 0 fraction 12 3.04 2.0 6.08 100 0 fraction 13 3.21 2.0 6.42 1000 fraction 14 2.85 2.0 5.70 100 0 fraction 15 2.52 2.0 5.04 99.46 0.54fraction 16 1.07 2.0 2.14 92.89 7.11 fraction 17 0.48 2.0 0.96 74.8125.19 fraction 18 0.33 2.0 0.66 49.32 50.68 fraction 19 0.30 2.0 0.6022.75 77.25 fraction 20 0.28 2.0 0.56 25.32 74.68 fraction 21 0.27 2.00.54 17.5 82.5 fraction 22 0.28 2.0 0.56 17.29 82.71 fraction 23 0.282.0 0.56 13.47 86.53 fraction 24 0.28 2.0 0.56 17.42 82.58 fraction 250.27 2.0 0.54 14.09 85.91 fraction 26 0.27 2.0 0.54 15.36 84.64 fraction27 0.24 2.0 0.48 11.57 88.43 fraction 28 0.23 2.0 0.46 13.01 86.99fraction 29 0.19 2.0 0.38 12.61 87.39 fraction 30 0.14 2.0 0.28 13.2986.71

The resolution of the monomer peak and the aggregate peak was determinedusing the program PeakFit (Seasolve Software Inc.) to be 0.70.

Example 24 Chromatography Conditions

-   Polypeptide: Anti-Ang2/VEGF antibody-   Exchange material: Poros 50HS-   Column: 11 mm internal diameter, 250 mm length, 23 ml volume-   Flow rate: 90 cm/h-   Equilibration solution: 40 mM sodium phosphate buffer, adjusted to    pH 5.0, 3 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 40 mM sodium phosphate buffer, adjusted to pH 5.0, 3    column volumes-   Elution solution: 40 mM sodium phosphate buffer, adjusted to pH 7.5-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 12 column volumes

The conditioned anion exchange eluate was applied to a chromatographycolumn containing the cation exchange chromatography material. After theloading step at a flow rate of 90 cm/h the column was washed with washsolution (3 column volume). The antibody was recovered with a lineargradient elution method, whereby the pH value was varied (increased)from 5.0 to 7.5.

The ¾ antibody “co-elutes” with the antibody monomer and two ¾ antibodypeaks are detected.

Example 25 Chromatography Conditions

-   Polypeptide: Anti-Ang2/VEGF antibody-   Exchange material: Poros 50HS-   Column: 11 mm internal diameter, 250 mm length, 23 ml volume-   Flow rate: 90 cm/h-   Equilibration solution: 40 mM sodium phosphate buffer, 5% PEG 4000,    adjusted to pH 5.0, 3 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 40 mM sodium phosphate buffer, 5% PEG 4000, adjusted    to pH 5.0, 3 column volumes-   Elution solution: 40 mM sodium phosphate buffer, 5% PEG 4000,    adjusted to pH 7.5-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 12 column volumes

The conditioned anion exchange eluate was applied to a chromatographycolumn containing the cation exchange chromatography material. After theloading step at a flow rate of 90 cm/h the column was washed with washsolution (3 column volume). The antibody was recovered with a lineargradient elution method, whereby the pH value was varied (increased)from 5.0 to 7.5.

Example 26 Chromatography Conditions

-   Polypeptide: Anti-Ang2/VEGF antibody-   Exchange material: Poros 50HS-   Column: 11 mm internal diameter, 250 mm length, 23 ml volume-   Flow rate: 90 cm/h-   Equilibration solution: 40 mM sodium phosphate buffer, adjusted to    pH 5.0, 3 column volumes-   Loading: 20 g protein/1 chromatography material-   Wash solution: 40 mM sodium phosphate buffer, 10% PEG 4000, 8%    glycine, adjusted to pH 5.0, 3 column volumes-   Elution solution: 40 mM sodium phosphate buffer, 10% PEG 4000, 8%    glycine, adjusted to pH 7.5-   Elution method: linear gradient from 0% (v/v) to 100% (v/v) elution    solution in 12 column volumes

The conditioned anion exchange eluate was applied to a chromatographycolumn containing the cation exchange chromatography material. After theloading step at a flow rate of 90 cm/h the column was washed with washsolution (3 column volume). The antibody was recovered with a lineargradient elution method, whereby the pH value was varied (increased)from 5.0 to 7.5.

In FIG. 6 the distribution of antibody in monomeric form, ¾ antibodyfragment and antibody in aggregated form over the collected fractions ofthe cation exchange chromatography of the anti-Ang2/VEGF antibody on thecation exchange chromatography material is shown. The antibody inmonomeric form and the antibody in aggregated form and the ¾ antibodyfragment are separated.

The invention claimed is:
 1. A method for producing an antibody of theIgG class in monomeric form comprising the following steps: applying afirst solution that optionally comprises poly(ethylene glycol) andsorbitol to a cation exchange chromatographic material, therebyequilibrating the material; applying a solution comprising an antibodyof the IgG class to the equilibrated cation exchange chromatographymaterial, thereby loading the chromatography material; applying asolution comprising poly (ethylene glycol) having a concentration ofabout 10% by weight and sorbitol having a concentration of from 5% to20% by weight to the cation exchange chromatographic material, therebyseparating the antibody of the IgG class in monomeric form from theantibody in aggregate form and obtaining the antibody in monomeric form.2. A method for producing an antibody of the IgG class preparation withreduced host cell protein content comprising the following steps:applying a first solution that optionally comprises poly(ethyleneglycol) and sorbitol to a cation exchange chromatographic material,thereby equilibrating the material; applying a solution comprising anantibody of the IgG class to the equilibrated cation exchangechromatography material, thereby loading the chromatography material;applying a solution comprising poly (ethylene glycol) having aconcentration of about 10% by weight and sorbitol having a concentrationof from 5% to 20% by weight to the cation exchange chromatographicmaterial, thereby separating the antibody of the IgG class in monomericform with reduced host cell protein content from the antibody inaggregate form and obtaining the antibody in monomeric form with reducedhost cell protein content.
 3. The method according to claim 1,characterized in that the poly (ethylene glycol) has a molecular weightof about 3,500 Da+/−20%.
 4. The method according to claim 1,characterized in that the monomeric antibody has a molecular weight offrom 100 kDa to 200 kDa.